Raina Khyati, Modak Kirti, Premkumar Chitra, Joshi Gaurav, Palani Dhavapriya, Nandy Krittika, Sivamani Yazhini, Velayudhan Shaji R, Thummer Rajkumar P
Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India.
Department of Hematology, Christian Medical College, Vellore, India.
Stem Cell Rev Rep. 2025 Apr;21(3):859-871. doi: 10.1007/s12015-024-10836-x. Epub 2025 Jan 4.
Undifferentiated embryonic cell transcription factor 1 (UTF1) is predominantly expressed in pluripotent stem cells and plays a vital role in embryonic development and pluripotency maintenance. Despite its established importance in murine models, the role of UTF1 on human induced pluripotent stem cells (iPSCs) has not been comprehensively studied.
This study utilized CRISPR/Cas9 gene editing to create UTF1 knockout in human fibroblasts and iPSCs. We employed episomal vectors for reprogramming UTF1 knockout fibroblasts into iPSCs and analyzed the effects of UTF1 depletion on cellular morphology, pluripotency, and viability through Western blotting, PCR, and flow cytometry. In addition, we integrated an shRNA that downregulated the expression of UTF1 for mechanistic studies to understand the impact of UTF1 depletion in iPSC pluripotency and differentiation.
UTF1 knockout resulted in significantly reduced reprogramming efficiency and increased spontaneous differentiation, indicating its crucial role in maintaining human iPSC identity and stability. In knockdown experiments, gradual loss of UTF1 led to change in cellular morphologies and decreased expression of core pluripotency markers OCT4 and SOX2. Interestingly, unlike complete UTF1 knockout, the gradual downregulation of UTF1 in iPSCs did not result in apoptosis, suggesting that the loss of pluripotency can occur independently of the apoptotic pathways.
UTF1 is essential for maintaining the pluripotency and viability of human iPSCs. Its depletion affects the fundamental properties of stem cells, underscoring the potential challenges in using UTF1-deficient cells for therapeutic applications. Future studies should explore the mechanistic pathways through which UTF1 controls pluripotency and differentiation, which could provide insights into improving iPSC stability for clinical applications.
未分化胚胎细胞转录因子1（UTF1）主要在多能干细胞中表达，在胚胎发育和多能性维持中起关键作用。尽管其在小鼠模型中的重要性已得到确立，但UTF1对人类诱导多能干细胞（iPSC）的作用尚未得到全面研究。
本研究利用CRISPR/Cas9基因编辑技术在人类成纤维细胞和iPSC中创建UTF1基因敲除。我们使用附加型载体将UTF1基因敲除的成纤维细胞重编程为iPSC，并通过蛋白质免疫印迹、聚合酶链反应和流式细胞术分析UTF1缺失对细胞形态、多能性和活力的影响。此外，我们整合了一个下调UTF1表达的短发夹RNA进行机制研究，以了解UTF1缺失对iPSC多能性和分化的影响。
UTF1基因敲除导致重编程效率显著降低和自发分化增加，表明其在维持人类iPSC特性和稳定性方面的关键作用。在敲低实验中，UTF1的逐渐缺失导致细胞形态改变以及核心多能性标志物OCT4和SOX2的表达降低。有趣的是，与完全敲除UTF1不同，iPSC中UTF1的逐渐下调并未导致细胞凋亡，这表明多能性的丧失可能独立于凋亡途径发生。
UTF1对于维持人类iPSC的多能性和活力至关重要。其缺失会影响干细胞的基本特性，突显了使用UTF1缺陷细胞进行治疗应用时潜在的挑战。未来的研究应探索UTF1控制多能性和分化的机制途径，这可能为提高iPSC在临床应用中的稳定性提供见解。
