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

合作研究：在人间充质干细胞培养扩增过程中维持能量稳态以保留生物特性

• 批准号: 1743426
• 负责人: Yan Li
• 金额: $ 55.3万
• 依托单位: Florida State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-15 至 2022-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1743426&HistoricalAwards=false
• 关键词: Collaborative; Research; Maintaining; Energy; Homeostasis

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).

PI：Ma、Teng 提案：1743426 人间充质干细胞 (hMSC) 在细胞疗法方面具有巨大潜力，并已在近 500 项临床试验中得到应用。目标领域包括心肌病、左心室功能障碍、糖尿病和移植物抗宿主病（GvHD）、类风湿性关节炎（RA）和多发性硬化症（MS）等免疫疾病。 hMSCs 从成年捐赠者中少量分离出来，并通过持续的连续培养扩增进行大规模生产用于临床试验。许多研究表明，持续培养会降低 hMSC 的效力和临床潜力，但导致效力降低的关键因素尚不清楚。 PI 获得的初步结果表明，与烟酰胺腺嘌呤二核苷酸 (NAD) 相关的代谢物相关的代谢和细胞信号传导可能在控制培养中 hMSC 的效力方面发挥核心作用。该项目的目标是研究长期扩增过程中 NAD 代谢发生的生物学变化，然后利用这些知识开发新的工程实践，以维持 NAD 水平，从而达到所需的临床效果。该项目将首先确定可行性和具体治疗策略，然后评估经过治疗的 hMSC 在治疗多发性硬化症 (MS) 实验性自身免疫性脑脊髓炎 (EAE) 小鼠模型中的治疗效果。这项研究的成果将是新的基础知识和工程实践，加速干细胞技术向临床应用的转化。该项目通过在干细胞生物学、细胞代谢、反应工程和工艺设计等跨学科研究环境中培训本科生和研究生，对劳动力发展产生更广泛的影响。这种跨学科培训对于培养细胞治疗行业的下一代生物工程师尤其重要。干细胞代谢网络和细胞稳态的分析将纳入PI教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。参与实验室已建立合作关系，在国内女性和少数民族学生的教育方面具有丰富的经验。该项目与 HBCU（历史上的黑人学院和大学）建立了合作关系，预计将对科学和工程领域代表性不足群体的学生招募和教育产生重大影响。人类间充质干细胞 (hMSC) 是超过一半的干细胞治疗中的首选细胞，也是全球经过最多临床测试的细胞。 hMSC 的生物制造需要体外扩增，这会导致治疗效力逐渐丧失，从而导致临床结果不一致。培养诱导的 hMSC 特性变化伴随着代谢变化和细胞稳态破坏，其特征是基础自噬减少、端粒磨损和衰老增加。 PI 实验室已发表的初步研究表明，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+ 增强在恢复高传代 hMSC 中 hMSC 稳态以及表型和功能特性方面的有效性。该项目将测试以下假设：1) 体外扩增导致代谢变化以及 NAD+ 浓度和 NAD+/NADH 比率逐渐下降； 2）NAD代谢的这些改变导致细胞稳态的破坏； 3) 在扩增过程中维持 NAD+ 水平可有效恢复线粒体电位和细胞稳态，从而保留临床相关的 hMSC 功能。了解扩增过程中 hMSC 表型变化的调控机制，以及实施代谢方法以最大限度地提高扩增产量，同时保留其治疗效力，将加速基于 hMSC 的疗法的临床应用转化。该项目的智力价值在于解决我们在维持 hMSC 稳态和功能特性的能量代谢知识方面的根本差距。这一差距目前阻碍了 hMSC 更广泛的临床转化。虽然代谢是细胞事件各个方面的基础，但人们对其在大规模扩增过程中调节 hMSC 稳态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留培养扩增的 hMSC 的治疗特性，从而解决 hMSC 生物制造中的重大技术障碍。重要的是，代谢方法是大规模 MSC 制造中的一种可行策略，因为它满足监管要求并消除与基因转染相关的安全问题。该提案独特且具有潜在变革性的方面是 1) hMSC 体外扩增导致细胞稳态崩溃的新概念，2) NAD+/NADH 代谢和 NAD+ 依赖性去乙酰化酶是维持细胞稳态的细胞能量传感器， 3) NAD+增强是在大规模生产中保留 hMSC 特性的可实施且有效的策略。 该项目将建立一种新颖的代谢策略，以解决基于 hMSC 的细胞治疗中限制进展的障碍，因此对治疗细胞的先进生物制造 (ABTC) 具有广泛的影响。

项目成果

Influence of Microenvironment on Mesenchymal Stem Cell Therapeutic Potency: From Planar Culture to Microcarriers

• DOI: 10.3389/fbioe.2020.00640
• 发表时间: 2020-06-24
• 期刊: FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
• 影响因子: 5.7
• 作者: Tsai, Ang-Chen;Jeske, Richard;Li, Yan
• 通讯作者: Li, Yan

Agitation in a microcarrier-based spinner flask bioreactor modulates homeostasis of human mesenchymal stem cells

• DOI: 10.1016/j.bej.2021.107947
• 发表时间: 2021-02-02
• 期刊: BIOCHEMICAL ENGINEERING JOURNAL
• 影响因子: 3.9
• 作者: Jeske, Richard;Lewis, Shaquille;Li, Yan
• 通讯作者: Li, Yan

Size-Dependent Cortical Compaction Induces Metabolic Adaptation in Mesenchymal Stem Cell Aggregates

• DOI: 10.1089/ten.tea.2018.0155
• 发表时间: 2019-04-01
• 期刊: TISSUE ENGINEERING PART A
• 影响因子: 4.1
• 作者: Bijonowski, Brent M.;Daraiseh, Susan, I;Ma, Teng
• 通讯作者: Ma, Teng

Metabolism in Human Mesenchymal Stromal Cells: A Missing Link Between hMSC Biomanufacturing and Therapy?

• DOI: 10.3389/fimmu.2019.00977
• 发表时间: 2019-05-08
• 期刊: FRONTIERS IN IMMUNOLOGY
• 影响因子: 7.3
• 作者: Yuan, Xuegang;Logan, Timothy M.;Ma, Teng
• 通讯作者: Ma, Teng