Induced Pluripotent Stem Cell (iPSC)-Based Mapping of Globin Expression Throughout Human Erythropoietic Development

Hematopoietic differentiation protocols using pluripotent stem cells mainly capture the primitive wave of hematopoietic development. We created a β-globin reporter iPSC line that allows for the mapping of β-globin expression throughout erythroid development. TALENs were used to target the β-globin locus in iPSCs where a promoterless GFP cassette was fused in frame to the first codon of the β-globin gene allowing for visualization of β-globin expression at single cell resolution via GFP expression. About 1% of cells exceeded the GFP detection threshold at the most mature stage of differentiation, suggesting that the several log-fold increase in β-globin transcripts seen at the population level as the cells progress through erythroid differentiation could be the result of high levels of β-globin transcription in a small fraction of cells. Single cell RNA sequencing of GFP- and GFP+ sorted fractions showed significantly greater levels of β-globin transcripts in the GFP+ fraction, validating our reporter line as a tool to visualize and enrich for β-globin expressing cells. Establishment of the developmental time frame of iPSC-derived erythroid cells is challenging as in vitro differentiation cultures lack the spatiotemporal separation of the different hematopoietic waves present in the embryo. Due to the possibility of multiple hematopoietic programs co-existing in one well, bulk expression analyses should be interpreted with caution when used to ascribe primitive or definitive characteristics to cell populations. We used single cell RNA sequencing to dissect the globin expression profiles of individual cells. The majority of the cells express a combination of ε, γ and β globins, indicative of a definitive yolk sac identity. As β-globin transcripts increased, we observed a decrease in ε-globin transcripts, indicating that primitive/embryonic characteristics are gradually lost as cells gain more definitive/adult features. Some cells showed a complete lack of ε-globin transcripts but robust expression of γ- and β-globins, suggesting the presence of cells with characteristics of the definitive, adult-repopulating wave of hematopoiesis