Characterizing and identifying hESCs and their derivatives by Raman imaging
Self-renewable pluripotent human embryonic stem cells (hESC) can readily differentiate into embryoid bodies (EBs) that contain virtually all cell types. A major technical hurdle in this field has been the lack of an effective yet non-invasive method for definitively discriminating among undifferentiated hESCs and their derivatives. This isolation is vital for subsequent cell-based therapies. Although several physical methods have been effective for purifying lineages that express cell-specific markers on their surfaces, those of many other highly specialized cells remain unknown. Conventional purification methods that rely on genetic manipulation or immunostaining are unsuitable for clinical use. Here we plan to use laser tweezers Raman spectroscopy of single cells to determine differences between individual human embryonic stem cells and their derivatives. This technique is based on the intrinsic biochemical signatures of cells obtained through non-destructive inelastic light scattering. It could become the basis for a novel, rapid and entirely non-destructive cell screening method that can accurately identify individual cells. Inelastic light scattering from specific molecular vibrations within a cell yields information on the biochemical composition of individual cells. Light from a laser is focused to a tight spot that covers the dimensions of a single cell. A small fraction of this laser light will interact with proteins, DNA, RNA, and other biomolecules in the cell, and lose parts of its energy to excite molecular vibrations. The outgoing (scattered) laser light is slightly red-shifted and contains distinct sharp peaks, that can be used to determine the relative distributions and concentrations of specific biochemicals within the cell. We found that by analyzing the strength of these peaks, different cells can be accurately distinguished. This technique is very fast, non-destructive, non-invasive, and does not require special sample preparation. It is important to note that the absence of external reagents (such as fluorescent labels) yields test cells that are neither destroyed/modified nor contaminated during the analysis. We expect our technique to be well suited for the future rapid analysis and isolation of hESCs, which is crucial for their use in clinical applications. It is also more accurate and specific than standard flow cytometry, the current gold standard for cellular diagnostics that is based on light scattering and fluorescence detection of a limited number of exogenous biomarkers. This grant will support important, necessary experiments that will define the spectroscopic signature of a wide range of hESC cells and their derivatives. These signatures will lay the foundation for commercially viable rapid cell sorting based on select peaks determined from these spectra.
Self-renewable pluripotent human embryonic stem cells (hESC) can readily differentiate into embryoid bodies (EBs) that contain virtually all cell types. A major technical hurdle in this field has been the lack of an effective yet non-invasive method for definitively discriminating among undifferentiated hESCs and their derivatives. This isolation is vital for subsequent cell-based therapies. Although physical methods such as magnetic bead sorting have been effective for purifying lineages that express cell-specific surface markers (e.g. CD34 for hematopoeitic cells), those of many other highly specialized cells remain unknown. For instance, all heart-restricted markers known are either cytoplasmic or nuclear. Conventional purification methods, which rely on genetic manipulation or immunostaining, are unsuitable for clinical use. With the research proposed here, we will demonstrate and establish an important new capability of rapid, non-invasive hESC identification. Rapid discrimination at the single cell level will permit the isolation of uncontaminated hESCs for use in cell-based therapies. This will remove a major clinical hurdle and rapidly benefit patients in need of hESC-based treatments. We anticipate to generate basic discoveries as well as patentable application-based technologies for licensing activities. Collectively, this will help establish the State of California as a pioneer in stem-cell based therapies and as a biotech hub, and provide its citizens with both academic and economic benefits. When the initial funding period ends and with the data generated from this proposal, we also expect to bring in federal monies via the NIH to California.