Figure 9.
Experiments showing the stimulatory effect of pooled cord blood cells growing at high cell densities in the upper inserts of transwells on the ALL3 cells in the lower wells at a LD at which they will not grow without stimulation. (A) Comparative growth of HD and LD ALL3 cells, and pooled CB MNCs and enriched CD34+ cells starting at a HD (4 × 105 cells/ml) in the upper inserts of transwells. Pooled enriched CB CD34+ cells starting at a HD (4 × 105 cells/ml) without GFs and with stimulation by 3 and 7 GFs in the upper inserts of transwells. The CD34+ cells grow faster and to higher cell densities (to ~9-10,000,000 cells/ml) than the MNCs when stimulated by 3 or 7 GFs (to ~1.4-2.4 × 105 cells/ml, and neither grew with no GFs. The HD ALL3 cells grew faster then the CB CD34+ cells, but the latter reached about double the HD ALL3 ultimate highest cell density a few days later. (B) Comparative growth of ALL3 cells at a LD (5000 cells/ml) in the lower wells of transwells when stimulated by the following cells growing in the upper inserts: Left Panel: LD (5000/ml) alone and HD ALL3 cells (3 × 105 cells/ml)); enriched CB CD34+ cells (3 × 105/ml) without GFs and with 3 or 7 GFs, and Right Panel: CB MNCs with and without the same 3 and 7 GFs. The CB MNCs grew slower and to lower maximum cell densities in the upper inserts than the enriched CD34+ cells (A), but the former were more stimulatory to the LD ALL3 cells which grew to >2 × 106 cells/ml in the lower wells when stimulated by the MNCs in the upper inserts with 7 GFs (B, right panel). As shown in the left panel of (C), the CD34+ cells with 3 GFs were more stimulatory to the ALL3 cells in the lower wells than the CD34+ cells with 7 GFs, probably because the latter were approaching saturation density sooner. Unexpectedly the CB CD34+ cells with no GFs which didn’t grow in the upper insert (A) had minimal stimulatory effect on the LD ALL3 cells until after day 16 when the ALL3 began to grow with a DTs of ~24 hr during the next 6 days (B, Left Panel). The CB MNCs without GFs may also have begun to stimulate growth of the ALL3 cells by day 16 (B, right panel). (C) Growth of CB MNCs and CD34+ cells with and without 7 GFs in upper inserts of transwells to test the HD CB cells’ ability to stimulate the growth of the LD ALL3 cells in lower wells. This was another pooled CB transwell experiment in which the CB CD34+ cells without GFs at a high starting cell density in the upper insert grew slowly in the upper inserts but the CB MNCs did not (left panel). With 7 GFs the HD CB CD34+ cells after an initial decline, grew faster but had a high death rate as many cells differentiated and died, while the CB MNC barely grew (right panel). (D) Stimulatory effect of these same CB CD34+ and MNCs with 7 GFs in the upper inserts on the LD ALL3 cells in the lower wells. Despite the failure of the MNCs to grow in the upper insert, both the MNCs and CD34+ cells stimulated the LD ALL3 cells to grow with a DT of ~26 hr. Note the earlier growth but the much higher death rate of the ALL3 cells stimulated by the HD CB CD34+ cells (left panel) than by the HD MNCs (right panel). (E) Stimulatory effect of HD CB MNCs with no, 3 and 8 GFs on the ALL3 cells starting at three different cell densities in the lower wells. As expected based on the other experiments, the CB MNCs had the greatest stimulatory effect on the LD ALL3 cells started at 0.5-1 × 104 cells/ml, and negligible effect on ALL3 cells started at 50,000 cells/ml since they grow well by themselves at this intermediate cell density. The GFs used in all these experiments have no stimulatory effect on the ALL3 cells themselves (see Figure 2), only on the CB cells in the upper inserts.