Unravelling the Molecular Regulation of Mesendodermal Differentiation in Human Embryonic Stem Cells

这项由生物技术、生化和生物工程项目颁发的NSF奖支持研究人类胚胎干细胞(HESCs)向中内胚层分化的分子事件，该研究方法结合了传统的假设驱动的工程分析和定量蛋白质组学。由转化生长因子-β和Wnt配体分别通过Smad2/3和β连环蛋白介导的信号转导在调节人胚胎干细胞命运方面发挥着重要作用。特别是，Smad2/3和β-catenin活性对于维持多能hESC状态以及向中内胚层分化(心脏、血液等临床相关细胞类型的前体)至关重要。然而，hESCs解释Smad2/3和β-catenin信号保持未分化或启动分化的确切机制在很大程度上仍不清楚。研究人员推测，尽管Smad2/3和β-catenin信号对维持多能性状态很重要，但在未分化的hESCs中，Smad2/3和β-catenin的活性受到多种机制的限制。此外，hESCs的中胚层分化与限制未分化hESCs中Smad2/3和β-catenin的机制失活以及伴随而来的Smad2/3和β-catenin活性的增加有关。这一假说将使用常规生化分析、靶向定量质谱仪以及对未分化的hESCs以及分化为中内胚层的细胞的全局蛋白表达和蛋白磷酸化的测量相结合的方法进行验证。拟议的实验将提供对维持hESCs的多潜能状态并触发中内胚层分化的生化网络的定量洞察。多能细胞可以为再生医学带来革命性的变化，并为药物评估提供一种产生功能细胞类型的手段。然而，多能细胞的临床应用仍处于初级阶段，这主要是由于缺乏有效的过程将其分化为所需的细胞类型。对影响hESC命运的分子途径的定量了解，将极大地有助于设计有效的过程将hESCs分化为临床相关的细胞类型。研究人员还将把他们的研究与从K-12到研究生教育的多个层次的科学和工程教育结合起来。一小时的讲座将在高中夏令营进行，旨在交流hESC科学和技术所涉及的复杂问题。拟议研究的概念和结果还将丰富两门研究生课程，这两门课程已经由私营部门和私营部门共同教授，目前正在由这些部门教授。除了这些课程，本科生还将参与研究。将通过在校园内向学生团体发表演讲来鼓励妇女和少数族裔学生参与。

This NSF award from the Biotechnology, Biochemical and Biomass Engineering program supports work to study the molecular events underlying the differentiation of human embryonic stem cells (hESCs) towards the mesendodermal lineages using an approach that integrates conventional hypothesis-driven engineering analyses and quantitative proteomics.Signaling mediated by the TGF-beta and Wnt ligands, through the Smad2/3 and beta catenin proteins respectively, plays an important role in regulating hESC fate. In particular, Smad2/3 and beta-catenin activity is essential for maintaining the pluripotent hESC state as well as during differentiation to mesendodermal lineages (precursors of clinically relevant cell types such as heart, blood). However, the exact mechanisms through which hESCs interpret Smad2/3 and beta-catenin signals to remain undifferentiated or initiate differentiation remain largely unknown. The investigators hypothesize that though Smad2/3 and beta-catenin signaling is important to maintain the pluripotent state, the activity of Smads and beta-catenin is restricted through multiple mechanisms in undifferentiated hESCs. Further, mesendodermal differentiation of hESCs is associated with inactivation of mechanisms that restrict Smad2/3 and beta-catenin in undifferentiated hESCs and a concomitant increase in Smad2/3 and beta catenin activity. This hypothesis will be tested using a combination of conventional biochemical analyses, targeted quantitative mass spectrometry and measurements of global protein expression and protein phosphorylation in undifferentiated hESCs as well as cells differentiating to the mesendodermal lineage. The proposed experiments will provide quantitative insight into the biochemical networks that maintain the pluripotent state of hESCs and trigger mesendodermal differentiation. Pluripotent cells can revolutionize regenerative medicine and provide a means to produce functional cell types for drug evaluation. However, clinical application of pluripotent cells is still in its infancy, largely due to the lack of efficient processes to differentiate them to desired cell types. A quantitative understanding of the molecular pathways influencing hESC fate, obtained through analyses such as those proposed in this project, will greatly help in designing efficient processes for differentiation of hESCs to clinically relevant cell types. The investigators will also integrate their research with science and engineering education at multiple levels, from K-12 to graduate education. A one-hour lecture that seeks to communicate the complex issues involved in hESC science and technology will be presented at high school camps. The concepts and findings of the proposed research will also enrich two graduate courses that have been developed by, and are currently being taught, by the PI and co-PI. In addition to these courses, undergraduate students will be involved in research. Participation of women and minority students will be encouraged through presentations to student groups on campus.