Table 1.

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

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).