of transplanted bone marrow cells, investigators showed that the number of spleen colonies varied linearly with dose and that the curve passed through the origin, suggesting that single cells were capable of forming individual colonies. However, because colony formation was rare relative to the numbers of transplanted cells, it was possible that undispersed clumps of two or more cells were the actual initiators.  A classic paper resolved this issue by exploiting rare, cytologically visible genome rearrangements generated by irradiation. Recipient mice were first irradiated to deplete bone marrow cells, and then they were irradiated a second time after transplantation to generate rare genome rearrangements in the transplanted cell population. Spleen colonies were then screened to find ones that carried genome rearrangements. How do you suppose this experiment distinguishes between colonization by single cells versus cellular aggregates?
22–9 It is possible to purify hematopoietic stem cells using a combination of antibodies directed against cellsurface targets. By removing cells that expressed surface markers characteristic of specific lineages such as B cells, granulocytes, myelomonocytic cells, and T cells, investigators generated a population of cells enriched for stem cells. They further enriched this population for putative stem cells by positively selecting for cells that expressed suspected stem-cell surface markers. Spleen colony formation in irradiated mice by these putative stem cells and the unfractionated bone marrow cells is shown in Figure Q22–3. Given that only about 1 in 10 cells lodges in the spleen, do these results support the idea that the enriched population consists mostly of hematopoietic stem cells? What additional information would you need to have to feel confident that the enriched cells are true stem cells? What proportion of bone marrow cells are hematopoietic stem cells?
22–10 Generation of induced pluripotent stem (iPS) cells was first accomplished using retroviral vectors to carry the OSKM (Oct4, Sox2, Klf4, and Myc) set of transcription regulators into cells. The efficiency of fibroblast reprogramming was typically low (0.01%), in part because large numbers of retroviruses must integrate to bring about reprogramming and each integration event carries with it the risk of inappropriately disrupting or activating a critical gene. In what other ways, or other forms, do you suppose you might deliver the OSKM transcription regulators so as to avoid these problems?
percent of ˜ cellsexpressing HPAP number of colonies cells (problem 22–9).
Figure Q22–2 percentage of labeled β cells in pancreatic islets of mice at different ages (problem 22–7). all mice were injected with a pulse of tamoxifen at 6 to 8 weeks of age and then stained for human placental 1 alkaline phosphatase (hpap) at various times afterward. error bars 101 102 103 104 105 106 represent standard deviations. number of cells injected
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