Stem and progenitor cell production, telomere length, and telomerase activity of CB CD34+ cells generated after repeated expansion-isolation procedures (“fractionated” LTC)
. | Input . | 1st isolation, wk 4 . | 2nd isolation, wk 8 . | 3rd isolation, wk 12 . | 4th isolation, wk 16 . | 5th isolation, wk 20 . |
|---|---|---|---|---|---|---|
| CD34+, × 103 | 100 | 380 | 1 820 | 6 550 | 30 100 | 144 500 |
| CD34+ /38/Lin−, × 103 | 11 | 72 | 400 | 1 560 | 7 600 | 6 700 |
| CFC, × 103 | 7.9 | 15.7 | 20.7 | 222.7 | 1 033.4 | 2 408.3 |
| CFU-GEMM, × 103 | 1.8 | 3.8 | 17 | 43.7 | 501.7 | 647.4 |
| LTC-1C, × 103 | 14.3 | 40 | 73.5 | 791 | 1 063.5 | 0.082 |
| Telomere length by flow-FISH, kb* | 9.1 | 9.9 | 9.3 | 9.3 | 9.3 | 7.25 |
| Telomerase activity, TPG units | 41.95 | 27.06 | 16.56 | 11.1 | 10.1 | 4.8 |
| Engraftment, no. injected mice/no. positive† | ||||||
| Primary, 3 × 105 CD34+ infused | 6/6 | 11/11 | 5/5 | 7/7 | 7/7 | 5/5 |
| Secondary | 2/2 | 3/3 | 2/2 | 3/3 | 2/2 | 0/1 |
| Tertiary‡ | 0/2 | 1/1 | 1/1 | 1/1 | 1/1 | ND |
| Frequency of SRCs in CD34+ cells | 1:35 270 | 1:10 605 | 1:6 955 | 1:5 263 | 1:1 716 | 1:83 892 |
| SRC/100 000 CD34+ cells§ | 2.8 | 9.4 | 14.4 | 19 | 58.3 | 1.2 |
. | Input . | 1st isolation, wk 4 . | 2nd isolation, wk 8 . | 3rd isolation, wk 12 . | 4th isolation, wk 16 . | 5th isolation, wk 20 . |
|---|---|---|---|---|---|---|
| CD34+, × 103 | 100 | 380 | 1 820 | 6 550 | 30 100 | 144 500 |
| CD34+ /38/Lin−, × 103 | 11 | 72 | 400 | 1 560 | 7 600 | 6 700 |
| CFC, × 103 | 7.9 | 15.7 | 20.7 | 222.7 | 1 033.4 | 2 408.3 |
| CFU-GEMM, × 103 | 1.8 | 3.8 | 17 | 43.7 | 501.7 | 647.4 |
| LTC-1C, × 103 | 14.3 | 40 | 73.5 | 791 | 1 063.5 | 0.082 |
| Telomere length by flow-FISH, kb* | 9.1 | 9.9 | 9.3 | 9.3 | 9.3 | 7.25 |
| Telomerase activity, TPG units | 41.95 | 27.06 | 16.56 | 11.1 | 10.1 | 4.8 |
| Engraftment, no. injected mice/no. positive† | ||||||
| Primary, 3 × 105 CD34+ infused | 6/6 | 11/11 | 5/5 | 7/7 | 7/7 | 5/5 |
| Secondary | 2/2 | 3/3 | 2/2 | 3/3 | 2/2 | 0/1 |
| Tertiary‡ | 0/2 | 1/1 | 1/1 | 1/1 | 1/1 | ND |
| Frequency of SRCs in CD34+ cells | 1:35 270 | 1:10 605 | 1:6 955 | 1:5 263 | 1:1 716 | 1:83 892 |
| SRC/100 000 CD34+ cells§ | 2.8 | 9.4 | 14.4 | 19 | 58.3 | 1.2 |
Mean of 4 different experiments.
The presence of at least 0.1% of human CD45+, CD71+, and GpA+ cells in the BM of NOD/SCID mice defined a positive engraftment.
In addition, mice were to be positive for human lymphoid (CD34[−]CD19/20+) and myeloid (CD45/71+CD15/66b+) engraftment (5 positive events each/20 000 assessed). For secondary and tertiary transplants 10 to 30 × 106 unseparated BM cells of the primary or the secondary mice were transplanted into secondary or tertiary sublethally irradiated NOD/SCID mice, which were killed 6 to 8 weeks later.
The SRC frequency in a population of cells was determined by injecting cohorts of mice with several dilutions of cells (LDA). The presence of at least 0.1% of human CD45+, CD71+, and GpA+ cells in the BM of NOD/SCID mice plus the presence of a human colony in 500 000 plated unseparated bone marrow cells) defined a positive engraftment. In addition all mice were found to have detectable human lymphoid (CD34[−]CD19/20+) and myeloid (CD45/71+CD15/66b+) engraftment (5 positive events each/20 000 assessed). The LDA results were synthesized as described in Figure 6. The SRC frequency was calculated from the proportions of negative mice in each cohort, using L-Calc T software program, as described in “Patients, materials, and methods.”