Paradigm for Optimizing Stem Cell Differentiation

干细胞的鉴定和表征通过提供一个体外系统来研究人类发育，从胚胎干细胞生命的最早阶段到成体干细胞后来器官和组织的形成和维持，已经彻底改变了发育生物学领域。干细胞也为组织工程应用提供了正常人类细胞的潜在来源。干细胞能够产生功能性体细胞类型，这些类型很难或不可能从原始来源收集或扩增，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结合使得从一致的克隆细胞来源产生大量细胞成为可能。这些工程组织可以作为发育模型和筛选药物疗效或毒性的直接应用，并有望作为替代体内受损或被破坏组织的活体治疗方法。阻碍将实验室干细胞研究的进展转化为用于开发工程组织产品的大量细胞的一个技术障碍是缺乏强大的培养系统，无法有效地引导干细胞通过多种祖细胞状态产生均匀的体细胞群体。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一问题，以确定群体异质性的起源，并预测生产高产纯度体细胞的最佳策略。作为一个模型系统，他们将研究人类诱导多能干细胞（iPSC）向表皮角质形成细胞的分化。在iPSC向角质形成细胞分化的各个阶段，细胞表达特异性的、特征明确的分子标记，有助于量化分化率参数，这些细胞中间产物可以稳定地分离和表征。此外，已经开发了多种将iPSCs分化为角质形成细胞的方案，允许在过程优化期间比较不同的分化方法。研究人员提出了模型开发的迭代策略，预测分化结果，预测的实验测试，并利用新获得的数据改进模型。智力优势：该项目将为优化干细胞命运选择建立一个新的范例。通过本项目的完成，研究人员将通过设计培养条件来调节干细胞、祖细胞和分化细胞的增殖和直接分化，从而更好地了解如何构建从干细胞产生体细胞的过程。此外，研究人员将讨论如何通过诱导不需要的细胞群体死亡或衰老来控制细胞群体的异质性。该项目的成果将直接影响从多能干细胞中开发用于药物和消费品测试的工程皮肤产品，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类型的体细胞。总之，这些活动将为实验室规模和工业规模的干细胞培养的设计原则提供见解。更广泛的影响：项目目标的完成将促进干细胞工程领域的进步，通过提高扩展未分化干细胞和将干细胞分化为所需谱系的效率，从而提高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级别的干细胞工程师，并将向K-12学生、K-12教师、本科生和公众提供有关干细胞科学和工程的技术、伦理和政治方面的推广。

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.