Figure 12.
Effect of BP CML MNCs or enriched CD34+ cells on stimulation of the LD ALL3 cells. These experiments were only possible using BP CML cells from occasional BP CML patients because the blasts from most patients with advanced BP disease blasts died too rapidly to obtain meaningful results. (A) Growth of BP CML MNC and enriched CD34+ cells in QBSF medium without and with 7 GFs. After a sharp decline the surviving CD34+ cells from a BP CML grew slowly in QBSF-60 at HD for about 12-14 days when stimulated with 7 GFs, but not without; the MNCs hardly grew at all. (B) Stimulatory activity of BP CML MNCs and CD34+ cells stimulated with 7 GFs in (A). The MNCs and CD34+ culture supernates were collected on day 16 to replace the media of freshly prepared LD ALL3 cells to see if any of the SNs would stimulate the LD ALL3 cells to proliferate, but none of the SN were stimulatory; only the CD34+ SN plus 7 GFs is shown. (C) Growth of BP CML enriched CD34+ blasts at HD from another BP CMP patient grown in QBSF-60. All the cells from the cultures without or with only 3 GFs died promptly but some surviving CD34+ blasts stimulated with 7 GFs grew very slowly for about 10 days (DT= ~147 h) and then died (left panel). None of the supernates from these BP CML HD CD34+ cultures was stimulatory to the LD ALL3 cells (right panel, C). (D) Right Panel: The lack of growth and rapid loss of viability of the enriched CD34+ blasts from another BP CML patient even when stimulated by 3 or 7 GFs. LD ALL3 cells (5000 cells/ml) also in the upper inserts of transwells were 50% non-viable by the day 2 and 100% by day 8. As a control, ALL3 cells at HD (4 × 105 cells/ml) grew well without any GFs in the upper inserts. Left Panel: Stimulatory effects of these cells in the upper inserts on the LD ALL3 cells in the lower wells. The HD ALL3 cells stimulated rapid growth of the LD cells for the first 8 days, but the cells then died quickly, probably because the HD media was exhausted; as expected the LD ALL3 cells in the upper inserts were not stimulatory. Unexpectedly, although the HD CML BP CD34+ cells didn’t grow at all in the upper inserts (D), they did stimulate some of the LD ALL3 cells to proliferate slowly beginning around days 8-10, albeit with a continuing high loss of viability. (E) Growth of LD and HD ALL3 cells without GFs and HD enriched CD34+ BP CML blasts from a patient with newly diagnosed, untreated BP CML in upper inserts of tranwells stimulated with 0, 3, and 7 GFs added on days 0, 4, and 6. Enriched CD34+ cells were obtained from a newly diagnosed untreated patient with BP CML with over 80,000 circulating blasts/mm3. The CD34+ blasts started at 4 × 105 cells/ml in the upper transwell inserts grew rapidly to over 14 and 10 million cells/ml respectively when stimulated by 7 and 3 GFs, and even the blasts without GFs grew slowly for 18 days. The left panel shows that as usual the LD ALL3 cells didn’t grow at all while the HD grew with a DT of 42 hr accompanied by a progressive increase in non-viable cells. (F) Growth of the LD ALL3 cells (5000 cells/ml) in lower wells in transwells stimulated by cells in upper inserts as shown. As usually observed, the HD ALL3 in the left panel stimulated the LD ALL3 cells to proliferate sooner than the BP CML CD34+ cells, but not to as high an ultimate cell peak.