Stem and progenitor cell production, telomere length, telomerase activity, and NOD/SCID engraftment of CB CD34+ cells generated after “continuous” stroma-free LTC
. | . | Time (wk) . | . | . | . | . | . | |||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
. | Input . | 2 . | 4 . | 8 . | 12 . | 16 . | 20 . | |||||
| CD34+, × 103 | 100 | 1 590 | 2 770 | 4 996 | 11 447 | 25 796 | 25 000 | |||||
| CFC, × 103 | 7.6 | 228 | 272 | 1 250 | 2 967 | 3 037.5 | 64.3 | |||||
| LTC-IC, × 103 | 10.7 | 10.2 | 531.5 | 1 082 | 189.5 | 0 | 0 | |||||
| Telomere length | ||||||||||||
| By flow-FISH, kb* | 9.1 | 9.9 | 10.1 | 9.8 | 9 | 7.9 | 6.8 | |||||
| By TRF, kb† | 9.9 | 9.8 | 10.1 | 9 | 9 | 9 | 7.8 | |||||
| Telomerase activity, TPG units | 41.95 | 60.92 | 27.06 | 14.235 | 4.75 | 4.795 | 2.67 | |||||
| Engraftment, no. of injected mice/no. of positive | ||||||||||||
| Primary, 3×105 CD34+ infused‡ | 6/6 | ND | 12/12 | 4/4 | 4/4 | ⅓ | 0/3 | |||||
| Secondary | ¾ | ND | 5/5 | 2/2 | 2/2 | 0/1 | ND | |||||
| Tertiary§ | 0/3 | ND | 2/2 | 1/1 | 1/1 | ND | ND | |||||
. | . | Time (wk) . | . | . | . | . | . | |||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
. | Input . | 2 . | 4 . | 8 . | 12 . | 16 . | 20 . | |||||
| CD34+, × 103 | 100 | 1 590 | 2 770 | 4 996 | 11 447 | 25 796 | 25 000 | |||||
| CFC, × 103 | 7.6 | 228 | 272 | 1 250 | 2 967 | 3 037.5 | 64.3 | |||||
| LTC-IC, × 103 | 10.7 | 10.2 | 531.5 | 1 082 | 189.5 | 0 | 0 | |||||
| Telomere length | ||||||||||||
| By flow-FISH, kb* | 9.1 | 9.9 | 10.1 | 9.8 | 9 | 7.9 | 6.8 | |||||
| By TRF, kb† | 9.9 | 9.8 | 10.1 | 9 | 9 | 9 | 7.8 | |||||
| Telomerase activity, TPG units | 41.95 | 60.92 | 27.06 | 14.235 | 4.75 | 4.795 | 2.67 | |||||
| Engraftment, no. of injected mice/no. of positive | ||||||||||||
| Primary, 3×105 CD34+ infused‡ | 6/6 | ND | 12/12 | 4/4 | 4/4 | ⅓ | 0/3 | |||||
| Secondary | ¾ | ND | 5/5 | 2/2 | 2/2 | 0/1 | ND | |||||
| Tertiary§ | 0/3 | ND | 2/2 | 1/1 | 1/1 | ND | ND | |||||
ND indicates not done.
Mean of 4 different experiments.
Mean of 2 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, that were sacrificed 6 to 8 weeks later.