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by Jane Yui, Choy-Pik Chiu, and Peter M. Lansdorp

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1 by Jane Yui, Choy-Pik Chiu, and Peter M. Lansdorp
Telomerase Activity in Candidate Stem Cells From Fetal Liver and Adult Bone Marrow by Jane Yui, Choy-Pik Chiu, and Peter M. Lansdorp Blood Volume 91(9): May 1, 1998 ©1998 by American Society of Hematology

2 Telomerase activity in candidate HSC before culture.
Telomerase activity in candidate HSC before culture. TRAP products were generated from 2 μL of CHAPS extract (1,000 cell equivalents) in the presence (+) or absence (−) of RNase A. TRAP products generated from sorted cells on day 0 from 4 different cadaveric marrow (lanes 1 to 8); lane 9, no extract; lane 10, 1 amol M2R8 standard. Arrow indicates the position of the 35-bp amplified internal control. Jane Yui et al. Blood 1998;91: ©1998 by American Society of Hematology

3 Telomerase activity in candidate HSC after 5 days of culture.
Telomerase activity in candidate HSC after 5 days of culture. TRAP products using CHAPS extracts of total viable cells derived by culturing purified candidate HSC from BM4 for 5 days in SCF (lanes 1 and 2), FL (lanes 3 and 4), IL-3 (lanes 5 and 6), SCF + FL (lanes 7 and 8), SCF + IL-3 (lanes 9 and 10), FL + IL-3 (lanes 11 and 12), and SCF + FL + IL-3 (lanes 13 and 14). TRAP products were resolved on a 15% polyacrylamide gel, dried, and exposed to film for 48 hours. The intensity of the signals was analyzed by a densitometer using ImageQuant program and normalized to that of the internal control. Cellular extracts from BM1 and BM3 yielded similar results. Jane Yui et al. Blood 1998;91: ©1998 by American Society of Hematology

4 Phenotypic analysis of candidate HSC after culture.
Phenotypic analysis of candidate HSC after culture. Candidate HSC were cultured for 5 days in (A) SCF, (B) FL, (C) IL-3, (D) SCF + FL, (E) SCF + IL-3, (F) FL + IL-3, and (G) SCF + FL + IL-3. After 5 days, cells were stained with antibodies against CD34, CD45RA, and CD71. Profiles shown were from events gated for low PI, low SCC, and high expression of CD34. In (G) boxes I, II, and III represent the windows used to sort for CD34+CD45RAloCD71lo cells, CD34+CD45RAloCD71hi cells, and CD34+CD45RAhiCD71hi cells, respectively. The same windows were applied to Fig 4 and Table 1. Jane Yui et al. Blood 1998;91: ©1998 by American Society of Hematology

5 Telomerase activity in subpopulations of cells present after 5 days in cultures of purified candidate HSC from adult marrow. Telomerase activity in subpopulations of cells present after 5 days in cultures of purified candidate HSC from adult marrow. See also Fig 3G. (A) CHAPS extracts from BM4-derived cells (lanes 1 to 10): CD34− fraction (lanes 1 and 2), CD34+fraction (lanes 3 and 4), CD34+CD45RAloCD71lo fraction (lanes 5 and 6), CD34+CD45RAloCD71hi fraction (lanes 7 and 8), and CD34+CD45RAhiCD71hi cells and (lanes 9 and 10); CHAPS extracts from BM1-derived cells (lanes 11 to 14): CD34− fraction (lanes 11 and 12) and CD34+ fraction (lanes 13 and 14). All extracts were generated from 1,000 cells. Jane Yui et al. Blood 1998;91: ©1998 by American Society of Hematology

6 Jane Yui et al. Blood 1998;91: ©1998 by American Society of Hematology

7 Jane Yui et al. Blood 1998;91: ©1998 by American Society of Hematology

8 Comparison of telomerase activity in freshly isolated CD34+CD38− cells from fetal liver and adult marrow. Comparison of telomerase activity in freshly isolated CD34+CD38− cells from fetal liver and adult marrow. TRAP assay performed on CHAPS extracts equivalent to 1,000 cells in the absence (−) and presence (+) of RNase A. CD34+CD38− cells from adult marrow (lanes 1 and 2) and fetal liver (lanes 3 and 4). Lysis buffer (LB) served as the negative control (lane 5), whereas 293 cells at the equivalent of 20 cells (lane 6) and 10 cells (lane 7) served as the positive controls. Jane Yui et al. Blood 1998;91: ©1998 by American Society of Hematology


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