Blood transfusion plays a vital role in modern medicine. However, availability is contingent on donated blood, and frequent shortages pose a significant healthcare challenge. Ex vivo manufacturing of red blood cells (RBCs) derived from universal donor O-negative pluripotent stem cells emerges as a solution, yet the high cost of recombinant cytokines required for ex vivo erythroid differentiation remains a major barrier. Erythropoietin (EPO) signaling through the EPO receptor is indispensable to RBC development, and EPO is one of the most expensive components in erythroid-promoting media. Here, we used design-build-test cycles to develop highly optimized small molecule-inducible EPO receptors (iEPORs) which were integrated at a variety of genomic loci using homology-directed repair genome editing. We found that integration of iEPOR at the endogenous EPOR locus in an induced pluripotent stem cell producer line enabled culture with small molecule to yield equivalent erythroid differentiation, transcriptomic changes, and hemoglobin production compared to cells cultured with EPO. Due to the dramatically lower cost of small molecules vs. recombinant cytokines, these efforts eliminate one of the most expensive elements of ex vivo culture media-EPO cytokine. Because dependence on cytokines is a common barrier to ex vivo cell production, these strategies could improve scalable manufacturing of a wide variety of clinically relevant cell types. More broadly, this work showcases how synthetic biology and genome editing may be combined to introduce precisely regulated and tunable behavior into cells, an advancement which will pave the way for increasingly sophisticated cell engineering strategies.

中文翻译：

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工程化诱导信号受体以实现无促红细胞生成素的红细胞生成

输血在现代医学中起着至关重要的作用。然而，血液供应情况取决于捐献的血液，而频繁的血液短缺给医疗保健带来了重大挑战。离体制造源自通用供体 O 阴性多能干细胞的红细胞 (RBC) 成为一种解决方案，但离体红细胞分化所需的重组细胞因子的高成本仍然是一个主要障碍。通过 EPO 受体的促红细胞生成素 (EPO) 信号传导对于红细胞发育是不可或缺的，并且 EPO 是促红细胞培养基中最昂贵的成分之一。在这里，我们使用设计-构建-测试循环开发高度优化的小分子诱导型 EPO 受体 (iEPOR)，使用同源定向修复基因组编辑将其整合到各种基因组位点。我们发现，与用 EPO 培养的细胞相比，iEPOR 整合到诱导多能干细胞生产系的内源 EPOR 位点上，使得用小分子培养能够产生相同的红细胞分化、转录组变化和血红蛋白产量。由于小分子的成本比重组细胞因子显着降低，这些努力消除了离体培养基中最昂贵的成分之一 - EPO 细胞因子。由于对细胞因子的依赖是离体细胞生产的常见障碍，因此这些策略可以改善多种临床相关细胞类型的可扩展生产。更广泛地说，这项工作展示了如何将合成生物学和基因组编辑结合起来，将精确调控和可调的行为引入细胞，这一进步将为日益复杂的细胞工程策略铺平道路。