Characterization of patient-derived HAX1W44X-iPSC. Generated HAX1W44X-iPSCs were compared with H9 ESCs and showed activation of their pluripotency network. (A) Sanger sequencing of HAX1W44X-iPSCs confirmed the c.130_131insA mutation leading to a stop codon in exon 2 of HAX1. (B) High expression pattern of alkaline phosphatase in ESCs was also detectable in generated iPSCs. Original magnification ×100; Naphthol/Fast Red Violet Solution. (C-D) Detection of pluripotency factors OCT4, SOX2, and NANOG by (C) immunohistochemistry (original magnification ×200) and (D) quantitative RT-PCR (n = 3, reference bar represents H9 ESC). (E) Flow cytometric quantification of ESC surface marker SSEA-4. (F) Karyotype analysis showed no chromosomal aberrations after reprogramming in HAX1W44X-iPSCs. (G) Upon undirected EB-based differentiation, scorecard assays demonstrated the capability of generated iPSCs to differentiate in all 3 germ layers. Pluripotency genes were strongly downregulated. EB, embryoid body.
