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Title

ENABLING TOOLS FOR SINGLE CELL ANALYSIS

Creator

Li, Zhaoxia

Date

2011-07, 2011-07

Description

Cells are basic functional units of life. A cell function is mediated by proteins and genes, whose distribution and expression level depends...
Show moreCells are basic functional units of life. A cell function is mediated by proteins and genes, whose distribution and expression level depends remarkably on the microenvironment. In the native environment, individual cells behave differently but communicate with surrounding cells. It is imperative to investigate proteins and genes at the single cell level in their native environment. The current representative single cell analysis methods, fluorescent techniques, are the most direct tools to study a single cell. The commonly used methods to measure protein and gene expression levels in single cells are on the basis of fluorescence labeling, such as fluorescence-activated cell sorting and live cell microscopy. They become more powerful when combine with the use of microfluidic devices. The disadvantages of these methods are, (1) their limited sensitivity doesn’t allow the detection of low-abundance proteins and genes; (2) they are unable to detect the cell-to-cell difference within a population; (3) the cell-sorting based method is lack of spatial resolution since the isolation of cells from the natural environment is required for analysis. To tackle these challenges, we established atomic force microscopy based approaches for in-situ gene and protein analysis on a target single live cell. The methods provide the spatial and quantitative information of cells in their native culture environment. They are effective and sensitive to detect low-abundant proteins and genes. In this thesis work, we developed a novel immunofluorescence assisted affinity mapping (IF-AM) method, in which immunofluorescence provides the guidance to locate a desired type of cell in a cell community for performing affinity mapping to quantify the local protein density at a high spatial resolution. Due to the ability of directly assessing proteins of individual cells, the IF-AM method is shown to be a sensitive tool for xiii resolving subtle differences in the local expression of membrane proteins even at low abundance. In the following work, we improved the accuracy of protein quantification by adapting the separation work based calculation rather than the previously used maximum adhesion force based calculation, and established a practical model to analyze the data systematically. We applied the methods to investigate the membrane proteins TRA-1-81 and E-cadherin on human embryonic stem cells. The heterogeneous distribution of TRA- 1-81 and the homogeneous distribution of E-cadherin as well as the quantitative measurement of the protein local abundance provided comprehensive information in understanding the strategy of hES cells to maintain the stemness during cell proliferation and to initiate the differentiation. An mRNA retrieval method was also developed to perform the gene expression analysis on a single cell of a desired type in a cell community. This was achieved by using a functionalized AFM tip as a bait to bind and retrieve mRNA from a desired single cell, followed by sensitive Quantitative Polymerase Chain Reaction (Q-PCR) analysis. The extraction of mRNA from live cells was performed with non/negligible damage to the cells. The established method here enabled the gene expression analysis of individual live cells at the original sites without disrupting the cell context. Thus the gene expression of a target cell and its surrounding cells can be analyzed in parallel, deriving concrete data for understanding the behavior of one cell in concert with that of the surrounding cells in the same or different cell population. The method was successfully applied in the study of side population cells in ovarian cancer cells. The methods developed in this thesis are versatile, and can be broadly applied to the study of different membrane proteins and genes of various cell types.
Ph.D. in Chemistry, July 2011
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