Lecture3 - Lecture 3. Surrogate markers to characterize...

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Lecture 3. Surrogate markers to characterize adult stem cells Lisheng WANG Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine
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How are adult stem cells characterized? The basic characterization of an adult stem cell are a single cell (clonal) that self-renews and generates differentiated cells. Clonal assay will remove doubts about contamination with other cell types. The most rigorous assessment of these characteristics is to prospectively purify a population of cells ( usually based on cell surface macromolecules ) , transplant a single cell into an acceptable host without any intervening in vitro culture, and observe self-renewal and tissue, organ, or lineage reconstitution. However, this type of in vivo reconstitution assay is not well defined for many types of adult stem cells because of technical difficulties.
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Many different types of stem cells are found in very small populations in the human body (for instance 1 in 100,000 cells in blood). Stem cells look just like any other cells under a microscope in the tissue where they reside. Identification of these rare type of cells (a process like to find a needle in a haystack) is based on stem cell "markers." These markers are macromolecules located either inside stem cells or on cell surface. Use of specific “markers” to identify and isolate adult stem cells
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Characterize adult stem cell based on intracellular markers (inside stem cells) Intracellular proteins (transcriptional factors, filament, etc) –not suitable for live cells Promoter- GFP (green fluorescent protein) - suitable for the study of differentiation (e.g. mesoderm promoter – GFP : during the differentiation of embryonic stem cells, appearance of green color indicate the emergence of mesoderm)
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Characterize adult stem cell based on surface markers In vivo and in vitro analyses of single adult stem cells Select and purify macromolecules that mark adult stem cells Generate specific antibodies against a given specific macromolecules Label the antibodies with fluorescent probe/enzyme/biotin Purify adult stem cells according to the expression of specific macromolecules that are recognized by specific antibodies
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A given macromolecule can be recognized by a specific antibody generated from an immunized animal Antibody IgG Source of image: http://www.accessexcellence.org/RC/VL/GG/antiBD_mol.html
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The specific antibody will be conjugated with a fluorescent probe, forming a specific fluorescent tag Image source : http://www.piercenet.com/Objects/view.cfm?type=Page&ID=412BAC2E-F0BC-4ABA-A79B-887E413910AB
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ght The fluorescent probe can be detected under UV light FITC Image source: http://www. hmds.org.uk/figures/mabs.jpg
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Lecture3 - Lecture 3. Surrogate markers to characterize...

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