Human RPA1^E240K iPSC exhibit telomere shortening and reduced hematopoietic potential. (A) Left panel: Healthy control iPSC (RPA1^WT) following CRISPR/Cas9–guided homozygous c.718G>A (p.E240K, as E240K) modification within endogenous RPA1 locus (RPA1^E240K) confirmed with Sanger analysis. Right panel: Illustration of iPSC monolayer-based differentiation to HPs and subsequently to erythroid and myeloid cell lineages. Day 10 HP cells were fluorescence-activated cell-sorted and cultured in erythroid or myeloid differentiation media for 14 days. In parallel, iPSC-derived HP were further cultured until day 21 to assess for expression of pan-hematopoietic markers. (B) Immunoblot analysis of RPA1 expression in RPA1^WT and RPA1^E240K iPSC whole-cell extracts with histone H3 as loading control. Telomere length in RPA1^WT passage 17 and RPA1^E240K passage 12 iPSCs (C) and iPSC-derived HP cells (D) using quantitative FISH. Graphs represent mean ± standard error of the mean (SEM) of 1 of 3 independent experiments (Student t test, ****P < .0001). (E) Decreased percentage of CD43⁺CD45⁺RPA1^E240K hematopoietic cells compared with RPA1^WT at days 16 and 21. Data represent mean ± SEM of 2 independent experiments (Student t test, *P = .03, **P = .0074). (F) Graphical representation of CD71⁺CD235⁺ erythroid cells from iPSC-derived erythroid cultures at day 14. Data represent mean ± SEM of 4 independent experiments (Student t test, ***P = .0002). (G) Plot representation of CD45⁺CD11b⁺ myeloid cells from iPSC cultures. Data represent mean ± SEM of 2 independent experiments (Student t test, **P = .0018). EPO, erythropoietin; IL-3, interleukin-3; GMCSF, granulocyte-macrophage colony-stimulating factor; GCSF, granulocyte colony-stimulating factor; SCF, stem cell factor; TPO, thrombopoietin.