Distribution of erythro-myeloid (EM) CFCs and pre-B/LAK cell potentials in the subfractions of CD34+ EB cells: CD45+ and CD34+ CD45− cell fractions and CD34+ CD31hi and CD34+ CD31lo cell fractions.
| Sorted CD34+ Subsets . | VEGF (10 ng/mL) . | Relative Numbers (% EB cells) . | EM CFC + LFFC/104 Sorted Cells . | EM CFC + LFFC/106 EB Cells . | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| E . | M . | n/mast . | Mix . | Total CFC (+V/−V)* . | LFFC (+V/−V) . | Total CFC (+V/−V) . | LFFC (+V/−V) . | |||
| 1) CD45+ CD45− | − + − + | 0.9 3.3 4.8 10.4 | 1.5 1.0 1.8 3.8 | 458.5 378.0 126.0 178.8 | 1.5 1.5 3.0 1.0 | 1.5 3.5 8.8 6.0 | 463.5 384.0 (0.8)* 139.5 189.5 (1.4) | 0.4† 0.1† (0.3) 3 1 (0.3) | 417.2 1267.2 (3.0) 669.6 1970.8 (2.9) | 0.3 0.3 (1.0) 10 10 (1.0) |
| 2)‡ CD45+CD45− | − + − + | 1.2 2.4 4.9 10.6 | 0.8 1.8 2.1 1.8 | 233.5 204.0 88.5 71.8 | 0.3 0 1.3 0.3 | 0.8 1.2 4.9 3.0 | 235.5 207.3 (0.9) 96.8 76.8 (0.8) | 0 0 4 2 (0.5) | 282.6 497.4 (1.8) 474.1 814.1 (1.7) | 0 0 20 25 (1.3) |
| 3) CD31hi CD31lo | − + − + | 1.2 4.8 2.6 5.9 | 4.0 5.0 17.3 24.3 | 33.5 60.3 227.5 314.8 | 0 0.8 5.3 11.3 | 1.5 3.8 12.8 12.0 | 39.0 69.8 (1.8) 264.0 362.3 (1.4) | 3 3 (1.0) 0.2 0.3 (1.5) | 46.8 334.8 (7.2) 686.4 2137.3 (3.1) | 4 15 (3.8) 0.5 2 (4.0) |
| 4) CD31hi CD31lo | − + − + | 2.4 5.2 0.4 2.5 | 1.3 1.3 31.5 21.8 | 24.0 34.3 344.8 495.0 | 1.8 3.3 8.8 11.8 | 2.3 0.5 20.8 18.8 | 29.5 39.3 (1.3) 405.8 547.3 (1.3) | 4 3 (0.8) 2 2 (1.0) | 70.8 204.1 (2.9) 162.3 1368.1 (8.4) | 10 14 (1.4) 0.8 5 (6.3) |
| (Average +V/−V) | (1.2) | (0.9) | (3.9) | (3.0) | ||||||
| Sorted CD34+ Subsets . | VEGF (10 ng/mL) . | Relative Numbers (% EB cells) . | EM CFC + LFFC/104 Sorted Cells . | EM CFC + LFFC/106 EB Cells . | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| E . | M . | n/mast . | Mix . | Total CFC (+V/−V)* . | LFFC (+V/−V) . | Total CFC (+V/−V) . | LFFC (+V/−V) . | |||
| 1) CD45+ CD45− | − + − + | 0.9 3.3 4.8 10.4 | 1.5 1.0 1.8 3.8 | 458.5 378.0 126.0 178.8 | 1.5 1.5 3.0 1.0 | 1.5 3.5 8.8 6.0 | 463.5 384.0 (0.8)* 139.5 189.5 (1.4) | 0.4† 0.1† (0.3) 3 1 (0.3) | 417.2 1267.2 (3.0) 669.6 1970.8 (2.9) | 0.3 0.3 (1.0) 10 10 (1.0) |
| 2)‡ CD45+CD45− | − + − + | 1.2 2.4 4.9 10.6 | 0.8 1.8 2.1 1.8 | 233.5 204.0 88.5 71.8 | 0.3 0 1.3 0.3 | 0.8 1.2 4.9 3.0 | 235.5 207.3 (0.9) 96.8 76.8 (0.8) | 0 0 4 2 (0.5) | 282.6 497.4 (1.8) 474.1 814.1 (1.7) | 0 0 20 25 (1.3) |
| 3) CD31hi CD31lo | − + − + | 1.2 4.8 2.6 5.9 | 4.0 5.0 17.3 24.3 | 33.5 60.3 227.5 314.8 | 0 0.8 5.3 11.3 | 1.5 3.8 12.8 12.0 | 39.0 69.8 (1.8) 264.0 362.3 (1.4) | 3 3 (1.0) 0.2 0.3 (1.5) | 46.8 334.8 (7.2) 686.4 2137.3 (3.1) | 4 15 (3.8) 0.5 2 (4.0) |
| 4) CD31hi CD31lo | − + − + | 2.4 5.2 0.4 2.5 | 1.3 1.3 31.5 21.8 | 24.0 34.3 344.8 495.0 | 1.8 3.3 8.8 11.8 | 2.3 0.5 20.8 18.8 | 29.5 39.3 (1.3) 405.8 547.3 (1.3) | 4 3 (0.8) 2 2 (1.0) | 70.8 204.1 (2.9) 162.3 1368.1 (8.4) | 10 14 (1.4) 0.8 5 (6.3) |
| (Average +V/−V) | (1.2) | (0.9) | (3.9) | (3.0) | ||||||
The CD45+ (R7 in Figures 1 and 3) and CD34+ CD45− (R4 in Figure 1) cells as well as the CD34+ CD31hi (R5 in Figures 1 and 3) and CD34+ CD31lo (R6 in Figures 1 and 3) cells were isolated from the E14-derived EBs cultured with 1.5 to 2 ng/mL BMP-4 with or without 10 ng/mL VEGF by FACS. The sorted cells were distributed in four 35 mm-dishes, and the number of all colony types was scored on day 9. At the same time, 0.5 − 4 × 104cells/well were plated on confluent cultures of OP9 cells in 6-well plates, and pre-B/LAK cell foci (LFFC) were scored as described in “Materials and methods.” The average colony numbers and foci numbers were adjusted to either 104 sorted cells or 106 EB cells.
Numbers in parentheses are ratios of total CFC or LFFC number with VEGF vs. that without VEGF (+V/−V).
These were B220+ CD19− LAK cells; no CD19+ pre-B cells were found.
BMP-4 concentration was 2 ng/mL.