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. 2016 Jun 1;6(6):1177–1230.

Figure 4.

Figure 4

Single cell cloning of the enriched CD34+ cord blood (CB) cells using 60-well single cell cloning plates. (A) Growth of enriched CD34+ cord blood cells in liquid culture (QBSF-60) starting at 400, 1000, 5000, & 9000 cells/ml without GF and with 7 or 8 GFs (KL, FLT3, TPO, G-CSF, GM-CSF, IL-3, IL-6, +/- EPO); MFE= maximum fold expansion. The cells didn’t grow at all without GFs except at the highest starting cell density of 9000 cells/ml when they grew slightly. When stimulated with 7 or 8 GFs the cells grew rapidly at all starting cell densities for 13 days, and, depending on the starting density, they underwent 154x to 2500x MFE after which they began to die rapidly as most of the cells matured. The cells usually grew slightly faster with the addition of EPO and sometimes to higher numbers, but also usually with a higher death rate (NV cells). (B) Growth or no growth of same CD34+ CB cells as in (A) without any GFs plotted on a different scale to show transient proliferation at a starting cell density of 9000 cells/ml but not at lower cell densities. The graph shows the initial rapid proliferation of the CD34+ CB cells starting at 9000 cells/ml without any GF with a DT of ~22 hr during the first 48 hr, which then slowed abruptly and almost all the cells were dead by day 15. The cells started at the LD without GFs died promptly. (C) Growth of highly enriched CD34+ CB cells in 60-well single cell cloning plates without GFs and with 7 or 8 GFs. CD34+ CB cells hardly grew at all without GFs as single or 2 cells, but with 7 or 8 GFs respectively 79 and 72% of single cells, 60-86% of 2 cells, and 100% of 3 cells continued growing to >100 cells within 6-10 days. (D) Growth of enriched CB CD34+ cells in 60-well single cell plates when stimulated by 8 GFs, and representative examples of proliferative fate of individual single cells. The growth of representative single or two CD34+ CB cells shown in (C) in the right panel with 8 GFs is shown here in more detail: 20/28 (72%) of single cells grew to >100 viable cells within 6-10 days (blue symbols) while one didn’t grow at all and 6 produced the numbers of cells shown in the (C) and then died (right Panel). Some of the 20 single cells that reached >100 cells kept growing after day 13 until they packed the small well while others began dying after ~10 days. Cell counts >100 or 200 cells/well are only rough estimates because of extreme crowding in the tiny wells.