ALBERT

Description: The β-thalassemia syndromes are the most prevalent genetic disorder globally, characterised by reduced or absent β-globin chain synthesis. HbE/β-thalassemia is a subtype of β-thalassemia with extremely high frequency in Asia. Studying molecular defects behind β-thalassemia is severely impeded by paucity of material from patients and lack of suitable cell lines. Approaches to derive erythroid cells from induced pluripotent stem cells (iPSCs) created from patients are confounded by poor levels of erythroid cell expansion, aberrant or incomplete erythroid differentiation and foetal/embryonic rather than adult globin expression. In this study we generate an immortalised erythroid cell line from peripheral blood stem cells of a HbE/β-thalassemia patient. Morphological analysis shows the cells are proerythroblasts with some early basophilic erythroblasts, with no change in morphology over time in culture. The line differentiates along the erythroid pathway to orthochromatic erythroblasts and reticulocytes. Importantly, unlike iPSCs, the line maintains the haemoglobin profile of the patient’s red blood cells. This is the first human cellular model for β-thalassemia providing a sustainable source of disease cells for studying underlying disease mechanisms and for use as drug screening platform, particularly for reagents designed to increase foetal haemoglobin expression as we have additionally demonstrated with hydroxyurea.

Description: Background Human induced pluripotent stem cells (hiPSCs) offer a renewable source of cells for the generation of hematopoietic cells for cell-based therapy, disease modeling, and drug screening. However, current serum/feeder-free differentiation protocols rely on the use of various cytokines, which makes the process very costly or the generation of embryoid bodies (EBs), which are labor-intensive and can cause heterogeneity during differentiation. Here, we report a simple feeder and serum-free monolayer protocol for efficient generation of iPSC-derived multipotent hematoendothelial progenitors (HEPs), which can further differentiate into endothelial and hematopoietic cells including erythroid and T lineages. Methods Formation of HEPs from iPSCs was initiated by inhibition of GSK3 signaling for 2 days followed by the addition of VEGF and FGF2 for 3 days. The HEPs were further induced toward mature endothelial cells (ECs) in an angiogenic condition and toward T cells by co-culturing with OP9-DL1 feeder cells. Endothelial-to-hematopoietic transition (EHT) of the HEPs was further promoted by supplementation with the TGF-β signaling inhibitor. Erythroid differentiation was performed by culturing the hematopoietic stem/progenitor cells (HSPCs) in a three-stage erythroid liquid culture system. Results Our protocol significantly enhanced the number of KDR+ CD34+ CD31+ HEPs on day 5 of differentiation. Further culture of HEPs in angiogenic conditions promoted the formation of mature ECs, which expressed CD34, CD31, CD144, vWF, and ICAM-1, and could exhibit the formation of vascular-like network and acetylated low-density lipoprotein (Ac-LDL) uptake. In addition, the HEPs were differentiated into CD8+ T lymphocytes, which could be expanded up to 34-fold upon TCR stimulation. Inhibition of TGF-β signaling at the HEP stage promoted EHT and yielded a large number of HSPCs expressing CD34 and CD43. Upon erythroid differentiation, these HSPCs were expanded up to 40-fold and displayed morphological changes following stages of erythroid development. Conclusion This protocol offers an efficient and simple approach for the generation of multipotent HEPs and could be adapted to generate desired blood cells in large numbers for applications in basic research including developmental study, disease modeling, and drug screening as well as in regenerative medicine.