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WIREs Dev Biol
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Quantification of epithelial cell proliferation, cell dynamics, and cell kinetics in vivo

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The measurement of cell proliferation in vivo is usually carried out by the examination of static measures. These comprise the mitotic index or labeling indices using incorporation of DNA synthesis markers such as bromodeoxyuridine or tritiated thymidine, or intrinsic markers, such as Ki67 and proliferative cell nuclear antigen (PCNA). But static measures only provide a ‘snapshot’ of cell proliferation. Rate measures, including double labeling methods and the metaphase arrest method, can actually measure cell production rates but they are far less utilized at present. Transit times and migration rates can also be measured using pulse and chase labeling or by following the transit of labeled cells through the tissue. Simple indices of cell division can easily be confounded by concomitant changes in the compartment size and many alleged markers of proliferation have serious shortcomings, as the markers may be involved in multiple aspects of cell regulation. The complexities of studying proliferation in vivo are illustrated here with a focus on the gastrointestinal tract. Some of these methods can help elucidate the role of the stem cells and their relationship to label retaining cells. WIREs Dev Biol 2017, 6:e274. doi: 10.1002/wdev.274

Demonstration of the greater number of mitotic figures that can be quantified if the whole intestinal crypt can be studied, rather than in a histological section. The asterisks represent mitotic figures within the crypt and demonstrate that only one of these would be seen in a histological section (the shaded area), while all five are seen in a squash preparation.
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The cell cycle, showing the readily identifiable phases of mitosis and DNA synthesis.
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Diagram of the various factors involved in the cell flux in the small intestinal proliferative system.
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A summary of the results of the misoprostol investigation, demonstrating that while the labeling index was not altered, the labeling per gastric gland was significantly increased, which demonstrates the profound effect of not accounting for concomitant changes in the denominator. The red text indicates derived values.
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The correlation between crypt area and mitotic activity. (Reprinted with permission from Ref . Copyright 1991 Elsevier Press)
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The grand tour of the human gastrointestinal tract showing the remarkable correlation between gland/crypt area and mitoses per proliferative unit. (Reprinted with permission from Ref . Copyright 1991 Elsevier Press)
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A device for the preparation of en face mounts of the mouse small intestine and colon. The segments are threaded over the stainless still rods and the top of the device acts as a cutting guide.
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Swiss roll of the proximal third of the mouse intestine. H & E staining.
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Microdissected small intestinal crypts showing one crypt in fission. Note the intensely staining.
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The crypt cycle, in which crypts can divide and increase their number.
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Diagram of the metaphase arrest method as applied to the intestinal crypts.
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Diagram of the gastric gland and a plot of the distribution of labeled cells within the gland. The black curve represents labeling at time 0 and the red curve a later labeling distribution. Calculating the change in the half maximum labeling values with time can be used to determine cell migration.
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Mouse small intestine labeled with two pulses of tritiated thymidine given 1 h and 17 h before sampling. The densely labeled cells represent the later pulse label. The difference in labeling is relatively easy to distinguish using autoradiography. Note the migration towards the lumen of the mitotic figures.
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Mouse small intestine stained with bromodeoxyuridine immunohistochemistry. The dense brown staining denotes the cells that incorporated BrdU in the S‐phase. See later for discussion of scoring thresholds as this section demonstrates a gradation in staining density and some background staining in the nonproliferative zones. Note the several mitotic figures and how they have moved into the crypt lumen.
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Autoradiograph of ovine ruminal epithelium showing the silver grains overlying the nucleus of a cell in the S‐phase. Semi‐thin H & E stained section.
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The principle of Tannock's factor to correct for the movement of mitotic figures into the lumen of the intestinal crypts when scoring histological sections.
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Illustration of the need to adhere to a rigorous definition of the stages of mitosis to be scored. In a good preparation more figures can be identified, whereas on a more indifferent preparation only the more readily identifiable figures will be seen.
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Section of the ovine ruminal epithelium showing two mitotic figures in the basal layer. Semi‐thin section stained with the Feulgen reaction, counter stained with light green.
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