Characterization and validation of the β-globin reporter human iPSC (hiPSC) line. (A) Targeting schematic depicting the introduction of a promoterless enhanced GFP (eGFP) cassette after the β-globin promoter. (B) Differentiation schematic and corresponding changes in β-globin transcript levels (quantitative reverse transcription PCR) during erythroid specification ± standard deviation (SD; 2 ≤ n ≤ 9). β-actin was used as a reference gene. CD34⁺ cells isolated from peripheral blood and specified toward the erythroid lineage (PB) were included as a primary control. (C-E) Characterization of wild-type (WT) or reporter (β_GFP) hiPSC-derived erythroid cells after 29 days of differentiation: representative FACS plots of GFP readout (C), bright field microscopy showing BFU-E colony morphology (×10) (D), and quantification of percentage of GFP⁺ cells ± SD (n = 9) (E). (F) HBB expression in individual GFP⁻ (gray) and GFP⁺ (green) cells based on scRNAseq counts. (G) Violin plot demonstrating a significant enrichment for HBB transcripts in the GFP⁺ fraction (green) compared with the GFP⁻ (gray) fraction based on normalized counts. ***FDR ≤ 0.0005, ****P ≤ .001. HA, homology arm; LCR, locus control region; PE, phycoerythrin; PGK, phosphoglycerate kinase promoter; PURO, puromycin resistance gene.