Single cell transcriptomic profiling provides insight into maturational and developmental status of pluripotent stem cell-derived erythroblasts

Robust β-globin expression in erythroid cells derived from induced pluripotent stem cells (iPSCs) would increase the resolution with which red blood cell disorders such as sickle cell disease and β thalassemia can be modeled in vitro. To better quantify efforts in augmenting β-globin expression, we report the creation of a β-globin reporter iPSC line through the insertion of a GFP cassette after the endogenous β-globin promoter, allowing for the mapping of β-globin expression throughout erythroid development in real time at single cell resolution. Sorting live GFP+ and GFP- cells at the most mature stage of erythroid differentiation, followed by single cell RNA sequencing (scRNAseq), identifies features that distinguish GFP- from GFP+ β-globin expressing cells and allows for the dissection of the developmental and maturational status of iPSC-derived erythroid cells. Co-expression of embryonic, fetal and adult globins in individual cells indicates a yolk sac erythro-myeloid progenitor (EMP) stage of hematopoietic development, representing the onset of definitive erythropoiesis. Within this developmental program, scRNAseq analysis identifies a gradient of erythroid maturation with GFP+ β-globin expressing cells showing increased maturation. In addition, scRNAseq analysis reveals that definitively patterned iPSC-derived erythroblasts resemble their postnatal counterparts in terms of gene expression and essential biological processes, confirming their potential for disease modeling and regenerative medicine applications. Single cell transcriptomic profiling of d29 iPSC-derived erythroid cells sorted for β-globin expression (GFP+ versus GFP-). The β-globin reporter iPSC line has an insertion of a GFP cassette after the endogenous β-globin promoter.