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

PI：Tzanakakis，Emmanouhl S. 提案编号：干细胞生物制造的发展对于实现干细胞和祖细胞用于再生医学治疗的潜力是必不可少的。提出了一个定量的框架，合理的设计和优化的干细胞的培养和转化为胰岛细胞的生物反应器，这是用于商业化生产的生物制药。所提出的方法考虑了干细胞群体的固有异质性，并可普遍应用于祖细胞分化成任何临床相关的表型。理论框架与功能性胰岛细胞的衍生作为实验模型系统相结合。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益处。定量模型对于合理设计和预测干细胞分化过程及其转化为生物制造实践是不可或缺的。同基因干细胞系固有的异质性使当前模型中假设的平均群体行为失效。该应用程序的中心假设，分化可以模拟人口平衡方程（PBE），这是多尺度的，并考虑到时间上不同的细胞内和细胞间的过程，包括随机事件影响干细胞生理。更重要的是，干细胞规格沿着一个特定的谱系可以描述的分布（分化）功能，没有许多现有的分化模型的限制性假设。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，在所提出的研究的第一个目标中，将通过成簇的规则间隔短回文重复序列（CRISPR）/Cas9基因组编辑来产生人类多能干细胞系。将报告基因插入多能性或胰腺内胚层命运采纳标志物的启动子下游。这些细胞系将有助于识别亚群和测量相关分布的解决方案的逆PBE问题，导致分化功能的提取。在第二个目标中，将从生物反应器培养物中确定描述自我更新干细胞状态的功能表达。干细胞的扩增和定向特化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其对维持干细胞多能性和特化的作用。在分化刺激物的不同组合和浓度的条件下，在搅拌的悬浮培养物中将人干细胞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培训提供极好的机会。胰腺细胞定向的预期定量景观将加速开发用于可规模化生产胰岛细胞的有效策略。所提出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（如心肌细胞、神经元、肝细胞和内皮细胞）方面的类似努力，对目前患有不治之症的患者的生活产生直接影响。

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.