CAREER: Enhancing Stem Cell Differentiation with CRISPR Activators via Non-integrating Methods

再生医学使用干细胞来替代受损组织，但这种方法需要具有已知活性的治疗细胞。目前还没有办法大量产生这样的细胞。在发育中的组织中，细胞分化涉及生长因子在特定时间的出现，以协调分化。这个项目建议评估使用病毒来激活基因，以复制这种协调。教育活动和实验室参观将为本科生和研究生设计。“干细胞与艺术”展示将突出干细胞研究的艺术、技术和伦理方面。它将为K-12学生、他们的家庭和普通大众开发。在这个项目中，人类诱导多能干细胞（iPSC）的分化将得到加强。特定于细胞系的基因将使用dCas9激活剂进行表观遗传学操作。协同激活介质（SAM）系统将用于在iPSCs中实现靶基因的激活。非整合腺相关病毒1 （AAV1）将向ipsc传递SAM激活剂。还将进行基因组级激活筛选，以鉴定促进内皮祖细胞（EP）分化的新基因。阳性候选细胞将用于增强与AAV1-SAM的EP分化。研究人员还将使用AAV-SAM促进β细胞分化。这些结果可能提供了令人信服的证据，表明利用SAM靶向基因激活可以用来增强iPSC的分化。除了小分子和生长因子外，这将为干细胞分化工具包引入一种新技术（SAM激活剂）。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Regenerative medicine uses stem cells to replace damaged tissues, but this approach requires therapeutic cells with known activity. There is currently no method to generate such cells in large quantities. In developing tissues, cell differentiation involves the appearance of growth factors at certain times to orchestrate differentiation. This project proposes to evaluate the use of viruses to activate genes in order to replicate that orchestration. Educational activities and lab tours will be designed to engage undergraduate and graduate students. A ‘‘Stem Cell and Art’’ showcase will highlight artistic, technical and ethical aspects of stem cell research. It will be developed for K-12 students, their families, and the general public.In this project, human induced pluripotent stem cell (iPSC) differentiation will be enhanced. Genes specific to cell lineages will be manipulated epigenetically using dCas9 activators. A Synergistic Activation Mediator (SAM) system will be used to achieve target gene activation in iPSCs. Non-integrating Adeno-associated virus 1 (AAV1) will deliver SAM activators to iPSCs. Genome-scale activation screens to identify novel genes that facilitate endothelial progenitor (EP) differentiation will also be performed. The positive candidates will be used to enhance EP differentiation with AAV1-SAM. The investigators will also use AAV-SAM to promote beta cell differentiation. These results may provide compelling evidence that targeted gene activation using SAM could be employed to enhance iPSC differentiation. This would introduce a novel technology (SAM activators) to the stem cell differentiation toolkit in addition to small molecules and growth factors.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.