Simple SummaryHuman pluripotent stem cells can differentiate into various tissues, making them an important source for cell therapy products. However, culturing pluripotent stem cells requires various growth factors, among which basic fibroblast growth factor is essential for maintaining stem cell ability. Unfortunately, basic fibroblast growth factor has a short half-life during cell culture and requires continuous supply, posing a significant challenge to culturing high-quality stem cells. To address this issue, we developed a thermostable basic fibroblast growth factor that is thermally stable and can maintain its activity for a longer period of time. We evaluated the various functions of human embryonic stem cells using thermostable basic fibroblast growth factor and found that cells cultured with thermostable basic fibroblast growth factor showed better proliferation, stemness, morphology, and differentiation. Thus, thermostable basic fibroblast growth factor with high heat resistance and persistence can play a critical role in securing high-quality stem cells during their cultivation.AbstractHuman embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can differentiate into various tissues and are an essential source of various disease models and therapeutics. Various growth factors are required in order to culture pluripotent stem cells, among which basic fibroblast growth factor (bFGF) is essential for maintaining stem cell ability. However, bFGF has a short half-life (8 h) under normal mammalian cell culture conditions, and its activity decreases after 72 h, posing a serious problem in the production of high-quality stem cells. Here, we evaluated the various functions of pluripotent stem cells (PSCs) by utilizing an engineered thermostable bFGF (TS-bFGF) that is thermally stable and maintains activity longer under mammalian culture conditions. PSCs cultured with TS-bFGF showed better proliferation, stemness, morphology, and differentiation than cells cultured with wild-type bFGF. In light of the importance of stem cells in a wide range of applications in the medical and biotechnology fields, we anticipate that TS-bFGF, as a thermostable and long-acting bFGF, can play a key role in securing high-quality stem cells through various sets of stem cell culture processes.