FMSG: Bio: Advancing Extracellular Vesicle Biomanufacturing of CRISPR-Edited Human iPSC-derived MSCs with Next-Generation Purification

由间充质干细胞（MSC）产生的细胞外囊泡（EV）因其多方面的治疗潜力而备受关注。然而，EV疗法的实际应用的巨大挑战是来自治疗性干细胞的EV的低产量。为了开发先进的EV生物制造工艺以满足临床需求，该项目旨在通过整合人诱导多能干细胞（hiPSC）以实现供体细胞来源的可扩展性，基因组工程以实现EV生物发生的可扩展性，以及先进的纳米膜技术以实现EV纯化的可扩展性，从而实现MSC衍生EV的规模化生产。此外，该项目旨在整合研究，教育，工业和多样性，重点是加强对学生多层次的研究。它将通过在两个校区开发新的合作课程，为研究生和本科生提供研究机会，并利用两个研究所的现有项目开展外联活动来实现。该项目不仅解决了用于治疗性EV生物制造的供体干细胞来源的可扩展性问题，还为EV生物学领域提供了新的见解，并为EV纯化瓶颈提供了新的解决方案。在此，这项研究汇集了一个跨学科的研究团队，以（1）产生具有EV生物发生和生物活性的合成增强剂的稳定的hiPSC系，（2）建立围产期组织样iMSC的无血清分化作为有效的EV工厂，（3）优化化学引发条件以增强细胞代谢活性以促进EV产生，以及（4）引入用于可规模化EV纯化的纳米袋膜技术，与其它标准技术相比，其具有改进的通量和产率。hiPSC技术、基因组工程和膜纳米纤维的整合为生产具有高度可扩展性、所需功能和临床相关性的治疗性EV提供了巨大的潜力，为EV生物制造的未来发展奠定了基础。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Extracellular vesicles (EVs) produced by mesenchymal stem cells (MSCs) have been highlighted for their multifaceted therapeutic potentials. However, a great challenge of practical applications of EV therapy is the low production of EVs from therapeutic stem cells. To develop an advanced EV biomanufacturing process to meet the clinical need, this project aims to enable scale-up production of MSC-derived EVs by integrating human induced pluripotent stem cells (hiPSCs) for scalability in donor cell source, genome engineering for scalability in EV biogenesis, and advanced nano-membrane technology for scalability in EV purification. Furthermore, this project is designed to integrate research, education, industry, and diversity with an emphasis on strengthening research exposure to the students at multiple levels. It will be achieved by developing new collaborative classes across two campuses, providing research opportunities to graduate and undergraduate students, and leveraging existing programs at both institutes for outreach activities. The close industrial collaboration will also present industry internship opportunities to the graduate students.This project not only addresses the scalable issues in donor stem cell sources for therapeutic EV biomanufacturing, but also provides new insights to the field of EV biology and new solutions to the bottleneck of EV purification. Herein, this research brings together an interdisciplinary research team to (1) generate a stable hiPSC line with synthetic boosters of EV biogenesis and bioactivity, (2) establish a serum-free differentiation of perinatal tissue-like iMSCs as a potent EV factory, (3) optimize chemical priming conditions to enhance cell metabolic activities for boosting the EV production, and (4) introduce the nanopocket membrane technique for scalable EV purification with improved throughput and yield, comparing to the other standard techniques. Integration of hiPSC technology, genome engineering and membrane nanofabrication offers great potential to produce therapeutic EVs with high scalability, desired functions, and clinical relevance for future advancement of EV biomanufacturing.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.