Huang Hui, Ye Kaiming, Jin Sha
Department of Biomedical Engineering Thomas J. Watson College of Engineering and Applied Science Binghamton University, State University of New York (SUNY), Binghamton, New York, USA.
Center of Biomanufacturing for Regenerative Medicine, Binghamton University, State University of New York (SUNY), Binghamton, New York, USA.
Biotechnol J. 2025 Apr;20(4):e70022. doi: 10.1002/biot.70022.
Human induced pluripotent stem cells (iPSCs) are an invaluable endless cell source for generating various therapeutic cells and tissues. However, their differentiation into specific cell lineages, such as definitive endoderm (DE) and pancreatic progenitor (PP), often suffers from poor reproducibility, due partially to their pluripotency. In this work, we investigated the impact of iPSC confluency during cell self-renewal and seeding density on cell metabolic activity, glycolysis to oxidative phosphorylation shift, and differentiation potential toward DE and PP lineages. Our findings demonstrated that cell seeding strategy influences cellular metabolic activity and the robustness of iPSC differentiation. iPSCs maintained at higher seeding density exhibited lower initial oxygen consumption rate (OCR) and metabolic activity. There is an optimal seeding density to ensure sufficient oxygen consumption during differentiation and to yield high expression of SOX17 in the DE lineage and high PDX1/NKX6.1 dual-positive cells in PPs. Interestingly, we found that cell confluency at the time of harvest has less impact on the efficacy of pancreatic lineage formation or metabolic activity. This study sheds light on the interplay between metabolic activity and iPSC lineage specification, offering new insights into the robustness of iPSC self-renewal and differentiation for creating human tissues.
人类诱导多能干细胞（iPSC）是用于生成各种治疗性细胞和组织的宝贵的无限细胞来源。然而，它们向特定细胞谱系的分化，如定形内胚层（DE）和胰腺祖细胞（PP），往往由于其多能性而重复性较差。在这项工作中，我们研究了iPSC在细胞自我更新过程中的汇合度以及接种密度对细胞代谢活性、糖酵解向氧化磷酸化转变以及向DE和PP谱系分化潜能的影响。我们的研究结果表明，细胞接种策略会影响细胞代谢活性和iPSC分化的稳健性。维持在较高接种密度的iPSC表现出较低的初始耗氧率（OCR）和代谢活性。存在一个最佳接种密度，以确保分化过程中有足够的氧消耗，并在DE谱系中产生高表达的SOX17以及在PP中产生高比例的PDX1/NKX6.1双阳性细胞。有趣的是，我们发现收获时的细胞汇合度对胰腺谱系形成的效率或代谢活性影响较小。这项研究揭示了代谢活性与iPSC谱系特化之间的相互作用，为iPSC自我更新和分化以创建人体组织的稳健性提供了新的见解。
