Collaborative Research: Maintaining Energy Homeostasis to Preserve Biological Properties during Culture Expansion of Human Mesenchymal Stem Cells

PI：MA，TengProposal：1743426人类间充质干细胞(HMSCs)在基于细胞的治疗方面具有巨大的潜力，已在近500项临床试验中得到应用。目标领域包括心肌病、左心功能不全、糖尿病和免疫疾病，如移植物抗宿主病(GvHD)、类风湿性关节炎(RA)和多发性硬化症(MS)。人骨髓间充质干细胞是从成人捐赠者中分离出来的少量干细胞，通过在培养中持续连续扩增的方式批量生产，用于临床试验。许多研究表明，持续培养会降低hMSCs的潜能和临床潜力，但导致这种潜能降低的关键因素尚不清楚。PI已获得初步结果，提示代谢和与烟酰胺腺嘌呤二核苷酸(NAD)相关的代谢物相关的细胞信号可能在控制hMSCs在培养中的效力方面发挥核心作用。该项目的目标是研究在长期扩张过程中NAD代谢的生物学变化，然后利用这些知识开发新的工程实践，以保持NAD的水平，从而达到预期的临床效果。该项目将首先确定可行性和具体的治疗策略，然后评估经治疗的hMSC对实验性自身免疫性脑脊髓炎(EAE)多发性硬化症(MS)小鼠模型的治疗效果。这项研究的结果将是新的基础知识和工程实践，加速将干细胞技术转化为临床应用。该项目通过在涵盖干细胞生物学、细胞新陈代谢、反应工程和过程设计的跨学科研究环境中培训本科生和研究生，对劳动力发展产生了更广泛的影响。这种跨学科的培训对于培养细胞治疗行业的下一代生物工程师尤为重要。干细胞代谢网络和细胞动态平衡的分析将被整合在PI讲授的高级化学反应工程课程中，以突出经典反应工程原理在干细胞工程中的应用。参与的实验室建立了合作关系，在国内女性和少数民族学生的教育方面经验丰富。该项目与HBCU(历史上的黑人学院和大学)建立了良好的关系，预计将对科学和工程专业中代表性不足的学生的招生和教育产生重大影响。人类间充质干细胞(HMSCs)是超过一半的干细胞疗法的选择细胞，也是全球经过临床测试最多的细胞。HMSCs的生物制造需要体外扩增，这会导致治疗效力的逐渐丧失，导致临床结果不一致。培养诱导的hMSC特性的改变伴随着代谢的改变和细胞内稳态的破坏，其特征是基础自噬减少，端粒磨损和衰老增加。PI实验室已发表的和初步的研究表明，在高传代的hMSCs中，NAD+浓度呈传代依赖性降低，其在维持细胞稳态中的关键作用，以及NAD+增强对恢复hMSC稳态以及表型和功能特性的有效性。该项目将检验以下假设：1)体外扩张导致NAD+浓度和NAD+/NADH比值的代谢变化和进行性下降；2)NAD代谢的这些变化导致细胞内稳态的破坏；3)在扩张过程中维持NAD+水平有效地恢复线粒体电位和细胞内稳态，从而保护临床相关的hMSC功能。了解hMSC在扩增过程中表型变化的调控机制，并实施代谢方法以最大限度地提高扩增产量，同时保持其治疗效力，将加速基于hMSC的治疗转化为临床应用。该项目的智力价值在于解决了我们在维持hMSC动态平衡和功能特性方面对能量代谢的认识上的根本差距。这一差距目前阻碍了hMSCs更广泛的临床翻译。虽然代谢是细胞事件的各个方面的基础，但人们对其在大规模扩增过程中调节hMSC动态平衡和表型的作用知之甚少。这项研究的结果将通过建立代谢策略来保护培养扩增的hMSCs的治疗特性，从而解决hMSC生物制造中的一个重要技术障碍。重要的是，代谢方法在大规模MSC制造中是一种可实施的策略，因为它符合监管要求，并消除了与基因转染相关的安全问题。这一建议的独特和潜在的变革性方面是：1)hMSC体外扩增导致细胞内稳态破坏的新概念；2)NAD+/NADH代谢和NAD+依赖的sirtuins是细胞能量感受器，维持细胞内稳态和特性；3)NAD+增强是在大规模生产中保持hMSC特性的一种可实施和有效的策略。该项目将建立一种新的代谢策略，以解决基于hMSC的细胞治疗中的进展限制障碍，因此在治疗细胞的先进生物制造(ABTC)中具有广泛的影响。

PI: Ma, TengProposal: 1743426Human mesenchymal stem cells (hMSCs) have tremendous potential for cell-based therapies and are featured in nearly 500 clinical trials. Targeted areas include cardiomyopathy, left ventricular dysfunction, diabetes and immune diseases such as graft-versus-host diseases (GvHD), rheumatoid arthritis (RA) and multiple schlerosis (MS). hMSCs are isolated in small numbers from adult donors and are mass-produced for clinical trials by sustained serial expansion in culture. Many studies have shown that sustained culture reduces the potency and clinical potential of hMSCs but the key factors that contribute to the reduced potency are not known. The PI has obtained preliminary results suggesting that metabolism and cell signaling associated with metabolites related to nicotinamide adenine dinucleotide (NAD) may play a central role in controlling the potency of hMSCs in culture. The objective of this project is to investigate the biological changes in NAD metabolism that occur during prolonged expansion, and then use this knowledge to develop new engineering practices that maintain the level of NAD and, therefore, the desired clinical effects. The project will first determine the feasibility and specific treatment strategy and then evaluate the therapeutic efficacy of the treated hMSC in treating an experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis (MS). The outcome of this research will be new fundamental knowledge and engineering practices that accelerate the translation of stem cell technology to clinical applications. The project has broader impacts on workforce development by training undergraduate and graduate students in a cross disciplinary research setting covering stem cell biology, cell metabolism, reaction engineering, and process design. This interdisciplinary training is especially important for developing the next generation of bioengineers in cell therapy industry. The analysis of metabolic networks and cellular homeostasis in stem cells will be integrated in the senior chemical reaction engineering course taught by the PI to highlight the application of classical reaction engineering principles in stem cell engineering. The participating laboratories have established collaborations, and are experienced in education of domestic female and minority students. The project, which has an established relationship with an HBCU (Historically Black Colleges and Universities), is expected to have a significant impact on the recruitment and education of students from underrepresented groups in science and engineering.Human mesenchymal stem cells (hMSCs) are the cell of choice in more than half of stem cell therapy and the most clinically-tested cells worldwide. Biomanufacturing of hMSCs requires in vitro expansion which leads to a gradual loss of therapeutic potency, contributing to inconsistent clinical results. The culture-induced changes in hMSC property are accompanied by metabolic shifts and a breakdown in cellular homeostasis as characterized by reduced basal autophagy, telomere attrition, and increased senescence. Published and preliminary studies from the PI's laboratory demonstrate a passage-dependent decrease in the NAD+ concentration, its key role in maintaining cellular homeostasis, and the effectiveness of NAD+-boosting to restore hMSC homeostasis andphenotypic and functional properties in high passage hMSCs. This project will test the hypotheses that: 1) in vitro expansion leads to a metabolic shift and a progressive decrease in NAD+concentration and the NAD+/NADH ratio; 2) these alterations in NAD metabolism lead to abreakdown in cellular homeostasis; and 3) maintaining NAD+ levels during expansion effectively restores mitochondrial potential and cellular homeostasis, thereby preserving the clinically relevant hMSC functions. Understanding the regulatory mechanisms underpinning the changes in hMSC phenotype during expansion and implementation of a metabolic approach to maximize expansion yield while preserving their therapeutic potency will accelerate the translation of hMSC-basedtherapy for clinical applications. The intellectual merit of the project lies in addressing fundamental gaps in our knowledge ofvenergy metabolism in maintaining hMSC homeostasis and functional properties. This gap currently prevents more widespread clinical translation of hMSCs. While metabolism underlies all aspects of cellular events, little is known about its role in regulating hMSC homeostasis and phenotype during large scale expansion. The results of this study will address a significant technological barrier in hMSC biomanufacturing by establishing a metabolic strategy to preserve the therapeutic properties of cultureexpanded hMSCs. Importantly, the metabolic approach is an implementable strategy in large scale MSC manufacturing because it meets the regulatory requirements and eliminates the safety concerns associated with gene transfection. The unique and potentially transformative aspects of this proposal are 1) the novel concept that the hMSC in vitro expansion leads to a breakdown in cellular homeostasis, 2)that NAD+/NADH metabolism and NAD+-dependent sirtuins are cellular energy sensors that preserve cellular homeostasis and properties, and that 3) NAD+-boosting is an implementable and effective strategy to preserve hMSC property in large scale manufacturing. The project will establish a novel metabolic strategy to address a progress-limiting barrier in hMSC-basedcell therapy and therefore has broad impacts in Advanced Biomanufacturing of Therapeutic Cells(ABTC).