Figure 5.
Single cell cloning with and without 7 GFs of enriched CD34+ blasts from transformed BP CML and AML patients. A and B. Growth in single cell cloning plates of total blasts and enriched CD34+ blasts obtained from a 32 year old woman with CP CML treated with Imatinib whose disease had undergone blast transformation after about a year. Her WBC was over 70,000/mm3 with 70% blasts. A. leukapheresis was done before resuming treatment and multiple aliquots of the total mononuclear cells (including all blasts) were frozen and later thawed for phenotyping and other studies. Flow Cytometry showed that only 6% of the blasts were CD34+ and these were included with the Total Blasts. Only 6% of the 70% PB blasts obtained by leukapheresis were CD34+ by flow cytometry and only ~0.1% of the CD34+ blasts were recovered after enrichment. Presumably the 6% CD34+ blasts were residual CP CML blasts. A. None of the single or 2 or 3 total blasts per well grew to more than 3-4 cells and most didn’t grow at all without GFs. They grew much better with 7 GFs: 15%, 39%, and 50% respectively of the cells starting at 1, 2, or 3 cells/well grew to >100 cells, and while 44% and 21% of the single and 2 cells didn’t grow at all, most of the wells starting with 3 cells/well that failed to reach >100 cells grew to at least 48-92 cells by Day 9. B. Growth of the 6% enriched CD34+ blasts from the total blasts in single cell cloning plates. Enriched CD34+ blasts and total blasts grew similarly when stimulated by 7 GFs with the latter slightly better. Respectively 11, 16, and 30% of the single, 2, and 3-4 enriched CD34+ blasts grew to >100 cells (solid color) compared to 15, 39, and 50% of the single, 2, and 3 total blasts. C and D. The patient (J.A.) from whom these blast cells were obtained by leukapheresis was a 42 year old man diagnosed with MDS which rapidly progressed to AML with a high number of circulating blasts. He was leukapheresed at another hospital and placed on hydroxyurea, but his WBC rose rapidly again. He had no other chemotherapy. On admission to Memorial Hospital the WBC was 71,000/mm3 with 84% blasts. Leukapheresis was repeated and numerous aliquots of the MNCs (mostly blasts) were frozen for later experiments. 30% of the blasts were CD34+ which were used for this experiment. The 7 GFs and their concentrations were the same as in previous experiments. C. Single cell cloning in QBSF-60 with and without 7 GFs of enriched CD34+ AML blasts from patients J.A. with AML progressed from MDS. All the blasts appeared viable and excluded trypan blue on day 0, but none of the single or few cells grew in single cell cloning experiments and all were dead after 2 days. D. Attempted growth of JA’s AML enriched CD34+ blasts in QBSF at HD (105 and 5 × 105 cells/ml) without and with 7 GFs. There was no growth at 105 or 5 × 105 cells/ml without GFs and the cells were all dead by 2 days. With 7 GFs after an initial sharp decline the surviving cells grew very slowly at starting densities of 105 and 5 × 105 cells/ml for 9 days with DTs of 120-135 hr, but almost all were dead by 2 weeks. The AML blasts were also cloned in methylcellulose, but no colonies were produced (not shown).