CFC depletion by antibodies against hematopoietic markers
. | CFC depletion, % . | . | . | . | |||
|---|---|---|---|---|---|---|---|
| Depleted cell subset, day of differentiation . | CFC-E . | CFC-Mix . | CFC-GM . | CFC-M . | |||
| CD34+ | |||||||
| 5 | 100 | NA | NA | NA | |||
| 6 | 96.8 ± 2.6 | 100 | 100 | 94.2 ± 11.6 | |||
| 7 | 62.3 ± 16.2 | 98.0 ± 3.5 | 97.7 ± 4.1 | 96.1 ± 3.6 | |||
| 8 | 37.8 ± 17.3 | 99.6 ± 0.8 | 99.1 ± 3.5 | 99.4 ± 2.3 | |||
| CD43+ | |||||||
| 5 | 98.3 ± 3.3 | NA | NA | NA | |||
| 6 | 100 | 100 | 99.9 ± 1.9 | 90.5 ± 13.2 | |||
| 7 | 100 | 99.3 ± 1.2 | 96.3 ± 5.0 | 96.4 ± 2.6 | |||
| 8 | 98.8 ± 1.4 | 100 | 97.3 ± 2.4 | 99.4 ± 1.0 | |||
| CD235a+ | |||||||
| 5 | 100 | NA | NA | NA | |||
| 6 | 100 | 69.7 ± 26.3 | 35.8 ± 22.6 | 38.1 ± 28.1 | |||
| 7 | 100 | 60.1 ± 7.6 | 19.0 ± 9.2 | 11.6 ± 8.3 | |||
| 8 | 99.0 ± 1.5 | 19.4 ± 7.7 | 5.2 ± 2.5 | 11.8 ± 5.4 | |||
| CD41a+ | |||||||
| 5 | 55.8 ± 18.0 | NA | NA | NA | |||
| 6 | 87.4 ± 9.6 | 20.8 ± 16.1 | 37.7 ± 23.7 | 35.5 ± 27.3 | |||
| 7 | 93.2 ± 9.3 | 31.6 ± 10.7 | 9.0 ± 5.6 | 12.7 ± 9.6 | |||
| 8 | 87.6 ± 13.3 | 4.1 ± 3.5 | 7.8 ± 6.6 | 11.3 ± 8.2 | |||
| CD45+ | |||||||
| 5 | <1 | NA | NA | NA | |||
| 6 | <1 | <1 | <1 | <1 | |||
| 7 | 4.7 ± 4.2 | 50.3 ± 6.5 | 46.2 ± 16.9 | 49.9 ± 17.5 | |||
| 8 | <1 | 87.4 ± 11.7 | 56.4 ± 13.1 | 75.0 ± 9.3 | |||
. | CFC depletion, % . | . | . | . | |||
|---|---|---|---|---|---|---|---|
| Depleted cell subset, day of differentiation . | CFC-E . | CFC-Mix . | CFC-GM . | CFC-M . | |||
| CD34+ | |||||||
| 5 | 100 | NA | NA | NA | |||
| 6 | 96.8 ± 2.6 | 100 | 100 | 94.2 ± 11.6 | |||
| 7 | 62.3 ± 16.2 | 98.0 ± 3.5 | 97.7 ± 4.1 | 96.1 ± 3.6 | |||
| 8 | 37.8 ± 17.3 | 99.6 ± 0.8 | 99.1 ± 3.5 | 99.4 ± 2.3 | |||
| CD43+ | |||||||
| 5 | 98.3 ± 3.3 | NA | NA | NA | |||
| 6 | 100 | 100 | 99.9 ± 1.9 | 90.5 ± 13.2 | |||
| 7 | 100 | 99.3 ± 1.2 | 96.3 ± 5.0 | 96.4 ± 2.6 | |||
| 8 | 98.8 ± 1.4 | 100 | 97.3 ± 2.4 | 99.4 ± 1.0 | |||
| CD235a+ | |||||||
| 5 | 100 | NA | NA | NA | |||
| 6 | 100 | 69.7 ± 26.3 | 35.8 ± 22.6 | 38.1 ± 28.1 | |||
| 7 | 100 | 60.1 ± 7.6 | 19.0 ± 9.2 | 11.6 ± 8.3 | |||
| 8 | 99.0 ± 1.5 | 19.4 ± 7.7 | 5.2 ± 2.5 | 11.8 ± 5.4 | |||
| CD41a+ | |||||||
| 5 | 55.8 ± 18.0 | NA | NA | NA | |||
| 6 | 87.4 ± 9.6 | 20.8 ± 16.1 | 37.7 ± 23.7 | 35.5 ± 27.3 | |||
| 7 | 93.2 ± 9.3 | 31.6 ± 10.7 | 9.0 ± 5.6 | 12.7 ± 9.6 | |||
| 8 | 87.6 ± 13.3 | 4.1 ± 3.5 | 7.8 ± 6.6 | 11.3 ± 8.2 | |||
| CD45+ | |||||||
| 5 | <1 | NA | NA | NA | |||
| 6 | <1 | <1 | <1 | <1 | |||
| 7 | 4.7 ± 4.2 | 50.3 ± 6.5 | 46.2 ± 16.9 | 49.9 ± 17.5 | |||
| 8 | <1 | 87.4 ± 11.7 | 56.4 ± 13.1 | 75.0 ± 9.3 | |||
Indicated cell subsets were depleted from hESC/OP9 cocultures by negative MACS technique using FITC/PE-conjugated specific mAbs and anti-FITC/PE secondary microbeads. Isotype-matched control mAbs were used for nonspecific depletion control. Depletion of cell subsets was in the range of 85% to 97% as determined by flow cytometry. MACS-processed cell samples were tested for erythroid (E), mixed (Mix), and myeloid (GM, M) CFCs by MethoCult GF+ assay. CFC depletion (%) was calculated by the following formula: 1 – (CFC counts in specific mAb-depleted sample – CFC counts in isotype control mAb-treated sample) × 100. Results are the mean ± SD of 4 independent experiments with H1 (n = 2) and H9 (n = 2) cells. By negative selection analysis, CFC depletion values (%) reflect the proportion of CFCs expressing a depletory marker. CD43 was the only cell marker expressed by all CFC types throughout the hESC/OP9 coculture.
NA indicates not applicable (no CFC detection in the isotype control mAb-treated samples).