Derivation and Pluripotency Validation of Six iPSC Lines From Amniotic Fluid Carrying Intermediate α‐Thalassemia Genotypes (‐‐ 3.7 /α SEA and ‐‐ 4.2 /α SEA )

Purpose Thalassemia intermedia (TI), or hemoglobin H (Hb H) disease, presents significant clinical heterogeneity, and its pathogenesis remains unclear. This study aimed to establish induced pluripotent stem cells (iPSCs) from amniotic fluid (AF) cells of fetuses diagnosed with intermediate α‐thalassemia genotypes (‐α 3.7 /‐‐ SEA and ‐α 4.2 /‐‐ SEA ) to provide a robust in vitro model for investigating disease mechanisms and exploring therapeutic strategies. Methods AF cells from six TI fetuses were reprogrammed using nonintegrating episomal vectors via electro‐transfection. The resulting iPSC lines underwent comprehensive characterization, including alkaline phosphatase (AP) staining, immunofluorescence (IF) for pluripotency markers (e.g., OCT‐4, SOX‐2, and TRA‐1‐60), quantitative reverse transcription real‐time PCR (RT‐qPCR) for the expression of pluripotency genes, G‐band karyotyping, and genetic analysis for thalassemia. Differentiation potential was assessed in vivo via teratoma formation in immunodeficient mice and in vitro through directed hematopoietic differentiation. Results Six stable iPSC lines were successfully established. All lines retained the progenitor’s intermediate α‐thalassemia genotype and exhibited normal karyotypes. They displayed typical embryonic stem cell (ESC)‐like morphology, were positive for AP, and expressed key pluripotency markers. In vivo, all lines robustly formed teratomas containing tissues derived from all three germ layers (endoderm, mesoderm, and ectoderm). Furthermore, in vitro hematopoietic differentiation protocols successfully induced the iPSCs to generate CD34 + /CD43 + hematopoietic progenitors, which were functionally validated by their ability to form hematopoietic colonies in colony‐forming unit (CFU) assays. Conclusion We have successfully derived and comprehensively characterized six patient‐specific iPSC lines from intermediate α‐thalassemia fetuses. These cell lines demonstrate robust pluripotency and maintain the capacity for hematopoietic differentiation. They represent a valuable resource for investigating TI pathophysiology, screening novel drugs, and developing future cell‐based therapeutic strategies.