Human pluripotent stem cells (hPSCs) can be used to investigate hematopoietic development and have the potential to advance cell-based therapies and to facilitate developmental biology studies. However, efficient ex vivo differentiation into hematopoietic lineages, including red blood cells (RBCs) of the erythroid lineage and immune cells such as macrophages of the myeloid lineage, is hampered by the need for precise temporal regulation of cytokines and growth factors.In this study, we developed an optimized protocol for hematopoietic lineage specification from hPSCs by fine-tuning the temporal dynamics of cytokine and growth factor applications. Prolonged mesodermal specification in the absence of hemogenic cytokines significantly enhanced the generation of hematopoietic progenitors (CD34＋ CD45＋ ) with robust functional potential. Early administration of interleukin (IL)-3 during hematopoietic specification promoted progenitor expansion and maturation. Supplementation of bone morphogenetic protein 4 at the hematopoietic maturation stage enhanced the differentiation efficiency and preferentially drove myeloid lineage commitment toward macrophages at the expense of erythroid differentiation. The timing of erythropoietin administration was important in erythroid lineage commitment, and delayed treatment (day 10) enhanced erythroblast expansion and RBC production. By contrast, the timing of IL-6, GM-CSF, and M-CSF exposure did not significantly affect macrophage differentiation efficiency, suggesting that myeloid lineage specification follows a default pathway under optimized differentiation conditions. These findings suggest a refined, time-controlled strategy for directing hematopoietic differentiation from hPSCs, and provide insight into therapeutic blood cell production, regenerative medicine, and ex vivo modeling of hematopoietic disorders.

Purpose: A relatively low response to chemotherapy has been reported for hormone receptor (HR)-positive breast cancer. In this study, we investigated the role of tryptophanyl-transfer RNA synthetase (WARS) in the chemotherapeutic response of HR-positive breast cancer. Methods: Pre-chemotherapeutic needle biopsy samples of 45 HR-positive breast cancer patients undergoing the same chemotherapeutic regimen were subjected to immunohistochemistry. To investigate the biological functions of WARS in HR-positive breast cancer, we conducted cell viability assay, flow cytometry analysis, caspase activity assay, Quantitative real-time polymerase chain reaction, and western blotting using WARS gene-modulated HR-positive breast cancer cells (T47D, ZR-75-1, and MCF7). Results: WARS overexpression in HR-positive breast cancer patients showed a significant correlation with favorable chemotherapy response. Downregulation of WARS increased cell viability following docetaxel treatment in tumor cell lines. On the other hand, WARS overexpression sensitized the therapeutic response to docetaxel. Additionally, downregulation of WARS caused a decrease in the number of apoptotic cell populations by docetaxel. Poly (ADP-ribose) polymerase cleavage and caspase 3/7 activity were increased in docetaxel-treated tumor cells with WARS overexpression. Conclusion Our results suggest that WARS might be a potential predictor for chemotherapy response in patients with HR-positive breast cancer as well as a novel molecular target to improve chemosensitivity.