1. Primitive quiescent leukemic cells from patients with chronic myeloid leukemia spontaneously initiate factor-independent growth in vitro in association with up-regulation of expression of interleukin-3
by Tessa L. Holyoake, Xiaoyan Jiang, Heather G. Jorgensen, Susan Graham, Michael J. Alcorn, Chris Laird, Allen C. Eaves, and Connie J. Eaves
Blood
Volume 97(3):
February 1, 2001
©2001 by American Society of Hematology

2. FACS profiles of the cell populations tested
FACS profiles of the cell populations tested.(A) Dot plot of CD34-FITC versus CD38-PE–labeled PI−cells showing CD34+38−, CD34+CD38+, and CD34− populations.
FACS profiles of the cell populations tested.(A) Dot plot of CD34-FITC versus CD38-PE–labeled PI−cells showing CD34+38−, CD34+CD38+, and CD34− populations. (B) Dot plot of Hst-stained versus Py-stained PI−CD34+ cells showing CD34+ G0 (R1) and CD34+ G1/S/G2/M (R2) populations. (C) Histogram of CFSE-stained PI−CD34+ cells showing TPO-resistant (M1) and TPO-responsive (M2) populations.
Tessa L. Holyoake et al. Blood 2001;97:
©2001 by American Society of Hematology

3. Distribution of clone sizes generated by different phenotypes of single CML cells cultured in growth factor–supplemented SFM.CD34+CD38− (▪), CD34+CD38+ (■), and CD34− (░) cells were cultured for 10 days in the presence of FL, SF, IL-3, IL-6, and G-CSF.
Distribution of clone sizes generated by different phenotypes of single CML cells cultured in growth factor–supplemented SFM.CD34+CD38− (▪), CD34+CD38+ (■), and CD34− (░) cells were cultured for 10 days in the presence of FL, SF, IL-3, IL-6, and G-CSF. The numbers on the abscissa indicate the minimum number of cells per clone for clones whose sizes ranged up to the next number shown. Results are from a representative experiment with cells from patient 2.
Tessa L. Holyoake et al. Blood 2001;97:
©2001 by American Society of Hematology

4. Phenotype analyses of cycling (G1/S/G2/M) and quiescent (G0) subsets of CD34+ cell populations.The numbers in the lower right quadrant of each plot indicate the percentage of PI− events of the corresponding phenotype (ie, positive for CD34 and negative for ...
Phenotype analyses of cycling (G1/S/G2/M) and quiescent (G0) subsets of CD34+ cell populations.The numbers in the lower right quadrant of each plot indicate the percentage of PI− events of the corresponding phenotype (ie, positive for CD34 and negative for CD38, CD45RA, CD71, or HLA-DR). Results are from FACS analyses of cells from patient 1.
Tessa L. Holyoake et al. Blood 2001;97:
©2001 by American Society of Hematology

5. Results showing that growth factor–independent G0 cells are exclusively BCR-ABL +.
Results showing that growth factor–independent G0 cells are exclusively BCR-ABL +. RT-PCR analysis ofBCR-ABL and actin expression was done on 20 clones derived from single, initially quiescent CD34+ cells (10 each from patients 1 and 2) after culture for 10 days in the absence of added growth factors. PCR products were hybridized by using complementary DNA probes specific for BCR-ABL and actin, respectively. Con 1, 1000 CML cells; con 2, 1000 normal bone marrow cells; con 3, 1000 CML cells without reverse transcriptase; con 4, water.
Tessa L. Holyoake et al. Blood 2001;97:
©2001 by American Society of Hematology

6. Clone sizes obtained in 10-day SFM cultures of single, initially quiescent (G0) CD34+ cells.Results from a representative experiment (patient 4) in the presence (▪) or absence (■) of added growth factors are shown.
Clone sizes obtained in 10-day SFM cultures of single, initially quiescent (G0) CD34+ cells.Results from a representative experiment (patient 4) in the presence (▪) or absence (■) of added growth factors are shown. The numbers on the abscissa indicate the minimum number of cells per clone for clones whose sizes range up to the next number shown. The difference in average clone size for clones generated in the presence and absence of added growth factors was significant (P < .001).
Tessa L. Holyoake et al. Blood 2001;97:
©2001 by American Society of Hematology

7. Results showing that quiescent CML cells rarely contain detectable levels of IL-3 or G-CSF transcripts.(A) RT-PCR detection of transcripts for BCR-ABL, IL-3, G-CSF, and actin (samples 3-5) or c-ABL (samples 7-9) in extracts of at least 10 initially quiescen...
Results showing that quiescent CML cells rarely contain detectable levels of IL-3 or G-CSF transcripts.(A) RT-PCR detection of transcripts for BCR-ABL, IL-3, G-CSF, and actin (samples 3-5) or c-ABL (samples 7-9) in extracts of at least 10 initially quiescent (G0) CD34+ cells or cycling (G1/S/G2/M [G1]) cells. (B) Single-cell RT-PCR analyses for IL-3 and G-CSF expression in initially quiescent (G0) CD34+ BCR-ABL + cells isolated from patients 3 to 5 and (C) for IL-3 expression in initially cycling (G1/S/G2/M) CD34+ BCR-ABL + cells isolated from patient 3 as a representative example confirming that more than 80% of such cells express IL-3.
Tessa L. Holyoake et al. Blood 2001;97:
©2001 by American Society of Hematology

8. Results showing that quiescent CML cells up-regulate IL-3 expression on entry into the cell cycle.(A) The histograms show the level of proliferation by single, initially quiescent (G0) CD34+ CML cells cultured for 4 days in SFM in the presence or absence of...
Results showing that quiescent CML cells up-regulate IL-3 expression on entry into the cell cycle.(A) The histograms show the level of proliferation by single, initially quiescent (G0) CD34+ CML cells cultured for 4 days in SFM in the presence or absence of added growth factors. The results shown are the mean level of proliferation for 288 single G0 cells analyzed from each of 3 similarly studied samples from patients with CML. (B) RT-PCR analyses of viable annexin-V−/PI− populations of G0or G1/S/G2/M CD34+ CML cells before (10 000 cells) and after (5000 cells) they were cultured for 4 days in either the absence (G0 −, G1 −) or presence (G0 +, G1 +) of added growth factors.
Tessa L. Holyoake et al. Blood 2001;97:
©2001 by American Society of Hematology