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.