22–5 In the 1950s, scientists fed 3H-thymidine to rats to label cells that were synthesizing DNA, and then followed the fates of labeled cells for periods of up to a year. They found three patterns of cell labeling in different tissues. Cells in some tissues such as neurons in the central nervous system and the retina did not get labeled. Muscle, kidney, and liver, by contrast, each showed a small number of labeled cells that retained their label, apparently without further division or loss. Finally, cells such as those in the squamous epithelia of the tongue and esophagus were labeled in fairly large numbers, with radioactive pairs of nuclei visible in 12 hours; however, the labeled cells disappeared over time. Which of these three patterns of labeling would you expect to see if the labeled cells were generated by stem cells? Explain your answer.
22–6 At any given time, intestinal crypts of mice comprise about 15 stem cells and 10 Paneth cells. After cell division, which occurs about once a day, the daughter cells remain stem cells only if they maintain contact with a Paneth cell. This constant competition for Paneth-cell contact raises the possibility that crypts might become monoclonal over time; that is, the crypt cells at one point in time might derive from only 1 of the 15 stem cells that existed at some earlier time. To test this possibility, you use the so-called confetti marker that upon activation expresses any one of three fluorescent proteins in the stem cells of the crypt. You then examine crypts at various times to determine whether they contain cells with multiple colors or only one color (Figure Q22–1). Do the crypts become monoclonal over time or not? How can you tell?
22–7 The origin of new β cells of the pancreas—from stem cells or from preexisting β cells—was not resolved until a decade ago, when the technique of lineage tracing was used to decide the issue. Using transgenic mice that expressed a tamoxifen-activated form of Cre recombinase under the control of the insulin promoter, which is active only in β cells, investigators could remove an inhibitory segment of DNA and thereby allow expression of human placental alkaline phosphatase (HPAP), which can be detected by histochemical staining. After a pulse of tamoxifen that converted about 30% of β cells in young mice to microvilli 22–8 One of the earliest assays for hematopoietic stem cells made use of their ability to form colonies in the spleens of heavily irradiated mice. By varying the amounts
Figure Q22–1 Fluorescent cells in crypts in mouse intestines at various times after activation of expression of fluorescent proteins (problem 22–6). the images are taken in the xz plane, which cuts through multiple crypts, as indicated in the schematic drawing. Roughly 50 crypts are visible in each section. Dotted white circles identify some individual crypts. Scale bars are 100 μm. (adapted from h.J. Snippert et al., Cell 143:134–144, 2010. with permission from elsevier.) cells that express HPAP, the investigators followed the percentage of labeled β cells for a year, during which time the total number of β cells in the pancreas increased by 6.5fold. How do you suppose the percentage of β cells would change over time if new β cells were derived from stem cells? What if new β cells were derived from preexisting β cells? Which hypothesis do the results in Figure Q22–2 support?