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

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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).