Recent studies have shown that retrovirus-mediated transfection with four transcription factors (Oct-3/4, Sox2, KLF4 and c-Myc) into mouse fibroblasts or mature B lymphocytes results in generation of induced pluripotent stem (iPS) cells. These iPS cells are similar to ESCs in morphology, proliferation, and pluripotency, as judged by teratoma formation and chimaera contribution. Thus they are promising donor sources for cell transplantation therapies. However, because it requires transformation of all four genes, the rate of successful generation of iPS cells is very low (~0.1%). Furthermore, viral gene delivery systems carry the inherent drawback of genetically modifying the host cell genome, potentially resulting in mutations that can lead to various genetic defects. To overcome these limitations, we will design and test a platform to reprogram individual mouse fibroblasts into iPS cells by directly injecting mRNAs using an integrated single cell manipulation system.
Analysis and manipulation of single cells is important for the understanding of cellular mechanisms of disease cause and progression, and for designing new treatment strategies. Individual cell manipulation has been gaining prominence in a wide range of applications, including cell sorting, gene and molecular delivery, cellular diagnostics, and single cell-based assays. Direct, physical cell manipulation offers much more precise selection and understanding of cell properties than data-averaging over a population of cells. Manipulation of cells is a challenging task, as it requires a precise, controllable manipulator system. The proposed integrated single cell manipulation system includes newly developed advanced tools that will capture, deliver, manipulate and detect cell functions at the single-cell level. One tool is a newly developed technology that enables capturing individual cells at fixed locations in a fabricated chamber with a mesh coupled to a negative pressure system. The other tool uses nanopipettes, glass pipettes that can be created with openings smaller than 50nm and can be used for delivery of reagents inside a cell, or the physical manipulation of cells. Nanopipettes can also be used for sensing of the intracellular environment by monitoring ionic current. We propose to further develop these technologies by integrating them to manipulate individual cells.
To test the application of the integrated technology, mouse fibroblasts will be injected with mRNAs in an attempt to generate iPS cells. If successful, injecting the mRNAs encoding the transcription factor cocktail may allow nuclear reprogramming of the differentiated cells into iPS without genetic modifying the cell, thus alleviating the deleterious effect of gene integration into chromosomes.
Statement of Benefit to California:
Advanced scientific exploration and collaboration: Automated single cell manipulation enables more effective stem cell research, generating important scientific data and refining the information gained from individual cells. The cabability of single-cell manipulation would open up extraordinary opportunities and collaborations that would lead to fundamental discoveries both in and beyond biomedical and biotechnology research, through interdisciplinary research at the cutting edge of stem cell biology and ingle cell genomics.
Accelerated Technology Development: In addition to projects involving the application of existing stem cells, many [REDACTED] stem cell scientists and engineers are developing new technologies and applications using current technology as a springboard. These scientists will be able to make significant contributions to methodological and technological improvements of existing technology, and [REDACTED] and California researchers will have the benefit of early access to these improvements. Newly developed tchnologies will likely be licensed to start-up or established local biotechnology companies, leading to additional jobs and tax revenue for the state of California.
Improved graduate student education: Graduate and undergraduate students being trained in the investigatorsí laboratories will be exposed to cutting edge technologies, which will enable them to explore related stem cell and biotechnology careers, ultimately leading to a better trained workforce for the state of California.
Improved funding and recruitment environment: The presence of the automated cell manipulation technologies will help attract additional outstanding faculty, postdoctoral fellows, and graduate students. Furthermore, the available tools will assist investigators in obtaining additional grant funding for projects, leveraging the CIRM and institutional investment. [REDACTED] will be better positioned to apply to several additional grant opportunities allowing faculty to firmly establish [REDACTED] as a leade in emerging stem cell and biotechnology advancements and increase the value of [REDACTED] as a CIRM partner.
This is a proposal to develop a technology for the manipulation of individual cells. This system will then be applied toward inducing pluripotency in mouse fibroblasts by injecting mRNAs encoding four reprogramming factors into single cells. To accomplish this, the applicant will develop a cell sifter that captures individual cells at fixed locations in a fabricated chamber, and an automated micromanipulator system that controls the movement of nanopipettes to align them with the captured cells. Reagents, such as mRNA, will then be delivered intracellularly through the nanopipettes. Five aims are proposed: (1) to design and optimize the nanopipette injection system; (2) to design and test the cell sifter for capturing single cells to fixed locations; (3) to design automated manipulation of nanopipettes synchronized with the cell sifter; (4) to reprogram mouse fibroblasts using the above system; and (5) to design analysis tools and methods for testing results of cell reprogramming.
Reviewers agreed that the potential impact of the proposed studies is high. The use of a nanopipette to inject molecules into individual cells, and being able to control the delivery temporally and the dose quantitatively, could enable more precise analyses of the kinetics and requirements for reprogramming than is currently possible using bulk cell populations. Furthermore, these studies could markedly improve nuclear reprogramming without having to genetically modify the cells.
Reviewers felt that the research plan holds significant promise, is supported by preliminary data and the milestones are well defined. However, reviewers identified serious weaknesses that diminished their enthusiasm. The nanopipette concept and cell sifter technology, a major focus of this proposal, are not new to the scientific community but they are new to this particular scientific application. One reviewer questioned whether cells needed to be captured to specific locations, since the nanopipette can be directed to the cells, and whether the proposed method for immobilization of the cells was appropriate. Another concern addressed the use of liposomes as a cellular model to work out the synchronization of the nanopipettes with the cell sifter. While the reviewers acknowledged the desire to employ a simplified system, they pointed out that liposomes lack the structural features of cells and that this would impact the effectiveness of the injection system. Reviewers commended the use of the single cell injection system to reprogram mouse fibroblasts, but felt this aim would be stronger if the applicants included information on how they might test dosage of each mRNA, which has been shown to affect reprogramming. Finally, reviewers felt that the last aim was ill guided; questioning that translation of exogenously introduced mRNA (as proposed) is going to be a specific indicator of reprogramming.
The applicant is an assistant professor with expertise in nanopipettes and molecular assay development, and a collaborator provides stem cell biology expertise. The budget was considered appropriate.
Although reviewers felt that the development of a single cell injection technology may yield promising applications, and that the applicant has the appropriate technological expertise, they were concerned that the proposed approach was not designed optimally, and that the underlying cell biology was not well thought through, diminishing their enthusiasm substantially.