Okamoto Masakazu, Inagaki Yusuke, Okamura Kensuke, Uchihara Yoshinobu, Saito Kenichiro, Kawai Akihito, Ogawa Munehiro, Kido Akira, Mori Eiichiro, Tanaka Yasuhito
Department of Rehabilitation Medicine, Nara Medical University, Kashihara, Nara, Japan.
Department of Orthopaedic Surgery, Nara Medical University, Kashihara, Nara, Japan.
Bone Rep. 2024 Nov 17;23:101816. doi: 10.1016/j.bonr.2024.101816. eCollection 2024 Dec.
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an essential tool for gene expression analysis; choosing appropriate reference genes for normalization is crucial to ensure data reliability. However, most studies on osteogenic differentiation have had limited success in identifying optimal reference genes. To the best of our knowledge, no optimal reference genes in three-dimensional (3D) osteogenic differentiation culture experiments using human induced pluripotent stem cells (hiPSCs) have been identified. In this study, we aimed to identify stable reference genes that could be used for normalization in gene expression analyses during the 3D osteogenic differentiation of hiPSCs using an atelocollagen sponge as a scaffold. Four algorithms-ΔCt, BestKeeper, NormFinder, and geNorm-were used to evaluate the stability of 14 candidate reference genes. Genes encoding TATA box-binding protein, hypoxanthine phosphoribosyltransferase 1, and 14-3-3 protein zeta polypeptide were identified as the most stable reference genes. In comparison, conventionally used reference genes (beta-2 microglobulin and beta-actin genes) ranked among those with low stability. We also demonstrated the successful 3D osteogenic differentiation of hiPSCs on atelocollagen sponge. Our findings provide valuable insights for reference gene selection and bone tissue regeneration from hiPSCs, which could improve the treatment prospects for bone defects and other similar conditions in regenerative medicine.
逆转录定量实时聚合酶链反应（RT-qPCR）是基因表达分析的重要工具；选择合适的内参基因进行标准化对于确保数据可靠性至关重要。然而，大多数关于成骨分化的研究在确定最佳内参基因方面成效有限。据我们所知，在使用人诱导多能干细胞（hiPSCs）进行的三维（3D）成骨分化培养实验中，尚未确定最佳内参基因。在本研究中，我们旨在确定稳定的内参基因，这些基因可用于在以去端肽胶原海绵为支架的hiPSCs三维成骨分化过程中的基因表达分析标准化。使用四种算法——ΔCt、BestKeeper、NormFinder和geNorm——来评估14个候选内参基因的稳定性。编码TATA盒结合蛋白、次黄嘌呤磷酸核糖基转移酶1和14-3-3蛋白ζ多肽的基因被确定为最稳定的内参基因。相比之下，传统使用的内参基因（β-2微球蛋白和β-肌动蛋白基因）在稳定性较低的基因中。我们还证明了hiPSCs在去端肽胶原海绵上成功进行了三维成骨分化。我们的研究结果为hiPSCs的内参基因选择和骨组织再生提供了有价值的见解，这可能改善再生医学中骨缺损和其他类似病症的治疗前景。
