Paradigm for Optimizing Stem Cell Differentiation

优化干细胞分化的范例

• 批准号: 1066311
• 负责人: Sean Palecek
• 金额: $ 33.99万
• 依托单位: University of Wisconsin-Madison
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2016-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1066311&HistoricalAwards=false
• 关键词: Paradigm; Optimizing; Stem; Cell; Differentiation

The identification and characterization of stem cells has revolutionized the field of developmental biology by providing an in vitro system to study human development, ranging from the earliest stages of life in embryonic stem cells to later organ and tissue formation and maintenance in adult stem cells. Stem cells also offer a potential source of normal, human cells for tissue engineering applications. Stem cells are able to generate functional somatic cell types that are difficult or impossible to collect or expand from primary sources, including neural cells, cardiac myocytes, and pancreatic beta-cells. The combination of high expansion potential and multipotency of stem cells permits generation of large numbers of cells from a consistent, clonal cell source. These engineered tissues have immediate applications as developmental models and in screening drug efficacy or toxicity, and possess future promise as living therapeutics to replace damaged or destroyed tissues in vivo.One technical hurdle impeding translation of advances in stem cell research in the laboratory to generation of large quantities of cells for developing engineered tissue products is the lack of robust culture systems that efficiently direct stem cells through multiple progenitor states to yield homogeneous populations of somatic cells. The investigators propose to address this issue by developing and implementing a stochastic model of cell fates to identify the origins of population heterogeneity and predict optimal strategies for producing somatic cells at high yield and purity. As a model system they will study human induced pluripotent stem cell (iPSC) differentiation to epidermal keratinocytes. Cells at various stages of iPSC differentiation to keratinocytes express specific, well-characterized molecular markers facilitating quantification of differentiation rate parameters and these cell intermediates can be stably isolated and characterized. Furthermore, multiple protocols for differentiating iPSCs to keratinocytes have been developed, permitting comparison of distinct differentiation methods during process optimization. The investigators propose an iterative strategy of model development, prediction of differentiation outcomes, experimental testing of predictions, and model improvement using newly-acquired data.Intellectual Merits: This project will establish a novel paradigm for optimization of stem cell fate choices. By completion of this project, investigators will better understand how to construct processes for producing somatic cells from stem cells by designing culture conditions to regulate expansion and direct differentiation of stem cells, progenitor cells, and differentiated cells. Furthermore, the investigators will address how to control cell population heterogeneity by inducing death or senescence in undesired cell populations. The project outcomes will have direct implications on developing engineered skin products from iPSCs for drug and consumer products testing, but the optimization methods devised here will also be generally applicable to producing any somatic cell type from any stem cell precursor. Together, these activities will provide insight into design principles for lab scale and industrial scale stem cell culture.Broader Impacts: Completion of the project objectives will facilitate advancement of the stem cell engineering field by improving the efficiency of expanding undifferentiated stem cells and differentiating stem cells to desired lineages, thereby enhancing the feasibility of translating advances in stem cell biology to development of in vitro tissues for screening and/or therapeutic applications. Education and outreach activities described in this proposal will also train stem cell engineers at the graduate and undergraduate levels, and will provide outreach to K-12 students, K-12 teachers, undergraduate students, and the general public on technical, ethical, and political aspects of stem cell science and engineering.

干细胞的鉴定和表征通过提供研究人类发育的体外系统而彻底改变了发育生物学领域，从胚胎干细胞的生命的最早阶段到成体干细胞的后期器官和组织形成和维持。 干细胞还为组织工程应用提供了正常人类细胞的潜在来源。 干细胞能够产生难以或不可能从主要来源收集或扩增的功能性体细胞类型，包括神经细胞、心肌细胞和胰腺β细胞。 干细胞的高扩增潜力和多能性的组合允许从一致的克隆细胞来源产生大量细胞。 这些工程化组织可立即用作发育模型和筛选药物功效或毒性，阻碍实验室中干细胞研究进展转化为产生大量细胞以开发工程组织产品的一个技术障碍是缺乏有效指导干细胞的强大培养系统通过多个祖细胞状态产生同质的体细胞群体。 研究人员建议通过开发和实施细胞命运的随机模型来解决这个问题，以确定群体异质性的起源，并预测以高产率和纯度生产体细胞的最佳策略。 作为一个模型系统，他们将研究人类诱导多能干细胞（iPSC）分化为表皮角质形成细胞。 处于iPSC分化为角质形成细胞的各个阶段的细胞表达特异性的、充分表征的分子标志物，其促进分化速率参数的定量，并且这些细胞中间体可以被稳定地分离和表征。 此外，已经开发了用于将iPSC分化为角质形成细胞的多种方案，允许在过程优化期间比较不同的分化方法。 研究人员提出了一种迭代策略的模型开发，预测分化的结果，实验测试的预测，并使用新获得的data.Intellectual优点模型改进：该项目将建立一个新的范例优化干细胞的命运选择。 通过完成该项目，研究人员将更好地了解如何通过设计培养条件来调节干细胞、祖细胞和分化细胞的扩增和直接分化，从而构建从干细胞生产体细胞的过程。 此外，研究人员将解决如何通过诱导不需要的细胞群死亡或衰老来控制细胞群异质性。 该项目的结果将对从iPSC开发用于药物和消费品测试的工程皮肤产品产生直接影响，但这里设计的优化方法也将普遍适用于从任何干细胞前体生产任何体细胞类型。 总之，这些活动将为实验室规模和工业规模干细胞培养的设计原则提供深入见解。项目目标的完成将通过提高扩增未分化干细胞和将干细胞分化为所需谱系的效率，促进干细胞工程领域的进步，从而增强了将干细胞生物学的进展转化为用于筛选和/或治疗应用的体外组织的开发的可行性。 本提案中描述的教育和推广活动还将培训研究生和本科生水平的干细胞工程师，并将向K-12学生，K-12教师，本科生和公众提供关于干细胞科学和工程的技术，伦理和政治方面的推广。