Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research, Singapore, Republic of Singapore.
Singapore Immunology Network, Agency for Science, Technology and Research, Singapore, Republic of Singapore.
Cell Prolif. 2022 Aug;55(8):e13218. doi: 10.1111/cpr.13218. Epub 2022 Mar 15.
Large-scale generation of universal red blood cells (RBCs) from O-negative (O-ve) human induced pluripotent stem cells (hiPSCs) holds the potential to alleviate worldwide shortages of blood and provide a safe and secure year-round supply. Mature RBCs and reticulocytes, the immature counterparts of RBCs generated during erythropoiesis, could also find important applications in research, for example in malaria parasite infection studies. However, one major challenge is the lack of a high-density culture platform for large-scale generation of RBCs in vitro.
We generated 10 O-ve hiPSC clones and evaluated their potential for mesoderm formation and erythroid differentiation. We then used a perfusion bioreactor system to perform studies with high-density cultures of erythroblasts in vitro.
Based on their tri-lineage (and specifically mesoderm) differentiation potential, we isolated six hiPSC clones capable of producing functional erythroblasts. Using the best performing clone, we demonstrated the small-scale generation of high-density cultures of erythroblasts in a perfusion bioreactor system. After process optimization, we were able to achieve a peak cell density of 34.7 million cells/ml with 92.2% viability in the stirred bioreactor. The cells expressed high levels of erythroblast markers, showed oxygen carrying capacity, and were able to undergo enucleation.
This study demonstrated a scalable platform for the production of functional RBCs from hiPSCs. The perfusion culture platform we describe here could pave the way for large volume-controlled bioreactor culture for the industrial generation of high cell density erythroblasts and RBCs.
从 O 型阴性（O-ve）人诱导多能干细胞（hiPSCs）大规模生成通用红细胞（RBCs），具有缓解全球血液短缺和提供安全、全年供应的潜力。成熟的 RBCs 和网织红细胞是红细胞生成过程中产生的不成熟 RBCs 的对应物，也可以在研究中找到重要的应用，例如在疟疾寄生虫感染研究中。然而，一个主要的挑战是缺乏大规模体外生成 RBCs 的高密度培养平台。
我们生成了 10 个 O-ve hiPSC 克隆，并评估了它们向中胚层形成和红细胞分化的潜能。然后，我们使用灌注生物反应器系统在体外进行高密培养红细胞的研究。
基于其三系（特别是中胚层）分化潜力，我们分离出了六个能够产生功能性红细胞的 hiPSC 克隆。使用表现最好的克隆，我们在灌注生物反应器系统中证明了小规模生成高密度培养的红细胞。经过工艺优化，我们能够在搅拌生物反应器中达到 3470 万细胞/ml 的峰值细胞密度，细胞活力为 92.2%。细胞表达高水平的红细胞标记物，具有携带氧气的能力，并能够去核。
本研究展示了从 hiPSCs 生产功能性 RBCs 的可扩展平台。我们在这里描述的灌注培养平台为大容量控制生物反应器培养铺平了道路，可用于工业规模生成高细胞密度的红细胞和 RBCs。
