Varga Eszter, Brandsma Eelke, Juarez-Garza Brenda E, Ramlal Renuka P E, Karrich Julien J, Laurent Adrien, Chavli Athina, Paskel Ruthmila, Fu Kerly, Flavell Richard A, von Lindern Marieke, Amsen Derk, Klijn Marieke E, van den Akker Emile
Department of Hematopoiesis, Sanquin Research Amsterdam, Amsterdam, 1066CX, The Netherlands.
Department of Biotechnology, Delft University of Technology, Delft, 2629HZ, The Netherlands.
Adv Sci (Weinh). 2025 Jul 12:e04725. doi: 10.1002/advs.202504725.
There is a constant worldwide need for blood products, traditionally obtained from donations. In vitro red blood cell (RBC) production could supplement this demand and offer benefits such as thorough screening for improved safety, the possibility of genetic manipulation to restore genetic deficiencies, and therapeutic loading. Induced pluripotent stem cells (iPSCs) are a promising cell source for transfusable RBCs due to their immortality and independence from donors. However, current iPSC differentiation protocols-including both monolayer and embryoid body-based systems-have failed to produce sufficient erythroid cells (10 per unit) for therapeutic application, primarily due to developmental immaturity, inefficient enucleation (5-25%), and suboptimal, static culture conditions lacking physiological relevance. This study describes the optimization of an iPSC to RBC differentiation platform and its step-by-step translation process to dynamic culture conditions, allowing scalability and eventual bioreactor application. The optimized dynamic culture yields ≈4.6 × 10 RBC/iPSC, requiring an estimated ≈4.9 × 10 iPSCs to produce a minitransfusion unit, achieving a consistent 40-70% enucleation rate and bona fide function, demonstrated by both in vitro and in vivo assays. Our feeder-free, GMP-compatible system accomplishes an enucleated RBC production rate sufficient for large-scale application and serves as a bridge to large-scale bioreactor RBC production, facilitating clinical application.
全球对血液制品一直有持续的需求，传统上血液制品是通过捐赠获得的。体外红细胞（RBC）生产可以补充这一需求，并带来诸多益处，如通过全面筛查提高安全性、进行基因操作以修复基因缺陷的可能性以及治疗性负载。诱导多能干细胞（iPSC）因其永生性和不依赖供体，是用于可输血红细胞的一种有前景的细胞来源。然而，目前的iPSC分化方案，包括基于单层培养和胚状体的系统，均未能产生足够数量（每单位10个）的红细胞用于治疗应用，主要原因是发育不成熟、去核效率低（5 - 25%）以及缺乏生理相关性的次优静态培养条件。本研究描述了iPSC到红细胞分化平台的优化及其向动态培养条件的逐步转化过程，从而实现可扩展性并最终应用于生物反应器。优化后的动态培养每iPSC可产生约4.6×10个红细胞，生产一个小型输血单位估计需要约4.9×10个iPSC，去核率达到40 - 70%且保持一致，体外和体内试验均证明其具有真正的功能。我们的无饲养层、符合GMP标准的系统实现了足以满足大规模应用的去核红细胞生产速率，并为大规模生物反应器红细胞生产搭建了桥梁，推动了临床应用。
