EAGER: Biomanufacturing: Development of a Quantitative Framework of Directed Stem Cell Differentiation in Scalable Bioreactors

Pi：Tzanakakis，Emmanouhl S.提案编号：1547785干细胞生物制造的发展对于实现干细胞和祖细胞在再生医学治疗中的潜力至关重要。提出了生物反应器中干细胞培养和转化为胰岛细胞的合理设计和优化的量化框架，用于生物药物的商业生产。所提出的方法考虑了干细胞群体固有的异质性，并可普遍应用于将祖细胞分化为任何临床相关的表型。理论框架与功能胰岛细胞的推导相结合，作为实验模型系统。预期的结果将解决胰岛替代疗法对细胞材料的未满足需求，对糖尿病患者的生活质量和相关的经济负担具有潜在的好处。定量模型对于合理设计和预测干细胞分化过程并将其转化为生物制造实践是不可或缺的。同基因干细胞群体固有的异质性使目前模型中假定的平均群体行为无效。这一应用的中心假设是，分化可以用群体平衡方程(PBE)来模拟，PBE是多尺度的，考虑了时间上不同的细胞内和细胞间过程，包括影响干细胞生理学的随机事件。更重要的是，沿着特定谱系的干细胞规格可以用分布(分化)函数来描述，而不需要现有分化模型的许多限制性假设。从细胞特征的可测量分布开始，研究人员建议通过PBE框架的逆解来提取分化函数。为此，在拟议研究的第一个目标中，人类多能干细胞系将通过成簇的规则间隔短回文重复序列(CRISPR)/Cas9基因组编辑来产生。报告基因将被插入多能性启动子下游或胰腺内胚层命运采纳标记。这些细胞系将有助于亚群的识别和相关分布的测量，以解决导致提取分化函数的PBE逆问题。在第二个目标中，描述自我更新干细胞状态的功能表达将从生物反应器培养中确定。干细胞的扩增和定向规范将在全自动搅拌悬浮生物反应器中进行，允许适当控制操作变量，并研究它们在维持干细胞多能性和规范方面的作用。在不同的分化刺激组合和浓度条件下，在搅拌悬浮培养中，人类干细胞将沿着胰腺内胚层谱系定向。计划中的研究活动将为本科生和研究生提供极好的培训机会。预期的胰岛细胞承诺的数量格局将加速开发可规模生产胰岛细胞的有效策略。建议的方法的普遍适用性将刺激在制造其他具有治疗意义的细胞类型方面的类似努力，如心肌细胞、神经元、肝细胞和内皮细胞，直接影响目前无法治愈的疾病患者的生活。

PI: Tzanakakis, Emmanouhl S. Proposal Number: 1547785The development of stem cell biomanufacturing is essential for realizing the potential of stem and progenitor cells for therapies in regenerative medicine. A quantitative framework is proposed for the rational design and optimization of the cultivation of stem cells and their conversion to pancreatic islet cells in bioreactors, which are utilized in the commercial production of biopharmaceuticals. The proposed approach takes into account the inherent heterogeneity of stem cell populations and can be universally applied to progenitor cell differentiation into any clinically relevant phenotype. The theoretical framework is combined with the derivation of functional islet cells as an experimental model system. The expected outcomes will address the unmet need for cellular material for islet replacement therapies with potential benefits to the quality of life of diabetes patients and associated economic burden.Quantitative models are indispensable for the rational design and prediction of stem cell differentiation processes and their translation to biomanufacturing practices. The inherent heterogeneity of isogenic stem cell ensembles invalidates the averaged population behavior assumed in current models. This application centers on the hypothesis that differentiation can be modeled by population balance equations (PBEs), which are multiscale and take into account temporally distinct intra- and intercellular processes, including stochastic events influencing stem cell physiology. More importantly, stem cell specification along a particular lineage can be described by a distribution (differentiation) function without many of the restrictive assumptions of existing differentiation models. Starting with measurable distributions of cellular traits, the investigators propose to extract differentiation functions via inverse solution of the PBE framework. For this purpose, in the first aim of the proposed studies, human pluripotent stem cell lines will be generated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. Reporter genes will be inserted downstream of promoters of pluripotency or pancreatic endoderm fate adoption markers. These cell lines will facilitate the identification of subpopulations and the measurement of relevant distributions for the solution of the inverse PBE problem leading to the extraction of differentiation functions. In the second aim, functional expressions describing the state of self-renewing stem cells will be determined from bioreactor cultures. The expansion and directed specification of stem cells will be carried out in a fully automated stirred-suspension bioreactor, permitting both the proper control of operational variables and the investigation of their role on the maintenance of stem cell pluripotency and on specification. Human stem cells will be directed along the pancreatic endoderm lineage in stirred suspension cultures under conditions of varying combinations and concentrations of differentiation stimuli. The planned research activities will provide excellent opportunities for undergraduate and graduate student training. The expected quantitative landscape of pancreatic cell commitment will expedite the development of efficient strategies for the scalable production of islet cells. The universal applicability of the proposed approach will spur similar efforts in the manufacturing of other therapeutically significant cell types such as cardiomyocytes, neurons, hepatocytes, and endothelial cells, with direct impact on the lives of patients afflicted by presently incurable diseases.