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

NSF生物技术、生物化学和生物质工程项目的这一奖项支持了利用整合了传统假设驱动的工程分析和定量蛋白质组学的方法研究人类胚胎干细胞（hESC）向中内胚层谱系分化的分子事件的工作。TGF-β和Wnt配体分别通过Smad 2/3和β连环蛋白介导的信号传导，在调控hESC命运中起重要作用。特别地，Smad 2/3和β-连环蛋白活性对于维持多能hESC状态以及在分化为中内胚层谱系（临床相关细胞类型如心脏、血液的前体）期间是必需的。然而，hESC解释Smad 2/3和β-连环蛋白信号以保持未分化或启动分化的确切机制在很大程度上仍然未知。 研究人员假设，尽管Smad 2/3和β-连环蛋白信号传导对维持多能状态很重要，但Smads和β-连环蛋白的活性在未分化的hESC中通过多种机制受到限制。此外，hESC的中内胚层分化与限制未分化hESC中Smad 2/3和β-连环蛋白的机制的失活以及Smad 2/3和β-连环蛋白活性的伴随增加相关。将使用常规生化分析、靶向定量质谱和未分化hESC以及分化为中内胚层谱系的细胞中的总体蛋白质表达和蛋白质磷酸化的测量的组合来测试该假设。拟议的实验将提供定量的生化网络，保持hESC的多能性状态，并触发中内胚层分化的见解。多能细胞可以彻底改变再生医学，并提供一种生产用于药物评估的功能细胞类型的方法。然而，多能细胞的临床应用仍处于起步阶段，主要是由于缺乏将它们分化为所需细胞类型的有效方法。 通过本项目中提出的分析获得的影响人胚胎干细胞命运的分子途径的定量理解，将大大有助于设计有效的人胚胎干细胞分化为临床相关细胞类型的过程。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相结合。一个小时的讲座，旨在沟通涉及hESC科学和技术的复杂问题将在高中营地提出。拟议研究的概念和发现还将丰富PI和联合PI开发并目前正在教授的两门研究生课程。 除了这些课程，本科生将参与研究。将通过在校园内向学生团体介绍情况来鼓励妇女和少数民族学生的参与。

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.