Induced Pluripotent Stem Cell (iPSC)-Based Mapping of Globin Expression throughout Human Erythropoietic Development
Authors: Vanuytsel Kim , Matte Taylor , Leung Amy , Naing Zaw Htut , Morrison Tasha A. , Chui David H.K. , Steinberg Martin H. , Murphy George J 
Published: April 2021

Abstract
It has recently become clear that in vitro hematopoietic differentiation protocols using pluripotent stem cells mainly capture the primitive wave of hematopoietic development with limited induction of the definitive wave that gives rise to cells with adult-type characteristics such as the ability to express adult β-globin. Recent efforts from our group in optimizing our original differentiation protocol to derive more definitively patterned erythroid cells from iPSCs have resulted in a significant increase in β-globin transcripts as well as robust β-globin protein expression at the most mature stage of differentiation. To better quantify our progress in augmenting β-globin expression and to track globin ontogeny in real-time, we created a β-globin reporter iPSC line that allows for the mapping of β-globin expression throughout erythroid development.
To create this tool, TALEN 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.
Interestingly, using our optimized protocol, only 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 just 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 in line with this hypothesis, thereby validating our reporter line as a tool to visualize and enrich for β-globin expressing cells.
Despite lacking β-globin protein expression, the GFP- cells were not completely devoid of β-globin transcripts. As recent studies suggest that translation is dynamically controlled in maturing red blood cells, our results might indicate that posttranscriptional mechanisms could impact the translation of these globin transcripts. In fact, mining of the genes differentially expressed between both populations revealed several transcripts enriched in the GFP+ fraction that code for proteins involved in the dynamic translational control of transcripts essential for maturing red blood cells. These findings suggest advanced maturation of the GFP+ fraction, as well as a role for posttranscriptional mechanisms in the regulation of β-globin protein expression during erythroid development from iPSCs.
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. Considering that the complexity of globin regulation in development might currently be underappreciated due to these factors, we also used single cell RNA sequencing to dissect the globin expression profiles of individual cells. Looking at the co-expression of different globins in individual cells, we found that the majority of the cells express a combination of embryonic (ε), fetal (γ) and adult (β) globins, indicative of a definitive yolk sac identity. Moreover, 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.
Further interrogation of the 'β-globin expression signature' that we distilled from the single cell RNA sequencing data will be instructive for future strategies aimed at increasing β-globin protein levels in iPSC-derived erythroid cells. Progress in this area will improve the resolution with which we can study hemoglobinopathies such as β-thalassemia and sickle cell disease and such strategies can then immediately be tested using the β-globin reporter iPSC line as a screening platform.

Disclosures
No relevant conflicts of interest to declare.