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

Robust ß-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 ß thalassemia can be modeled in vitro. To better quantify efforts in augmenting ß-globin expression, we report the creation of a ß-globin reporter iPSC line through the insertion of a GFP cassette after the endogenous ß-globin promoter, allowing for the mapping of ß-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+ ß-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+ ß-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. Overall design: Single cell transcriptomic profiling of d29 iPSC-derived erythroid cells sorted for ß-globin expression (GFP+ versus GFP-). The ß-globin reporter iPSC line has an insertion of a GFP cassette after the endogenous ß-globin promoter.