LAK Cells and Pre-B Cells Were Generated Specifically From CD34+ EB Cells
| Plated Cell No./Well . | Experiment 1* . | Experiment 2† . | Experiment 3† . | Experiment 4† . | |||
|---|---|---|---|---|---|---|---|
| IL-2 + IL-7 . | IL-2 . | IL-7 . | IL-2 . | IL-7 . | IL-2 . | IL-7 . | |
| CD34+ | |||||||
| 105 | LAK‡ + 1-153/(pre-B+)1-155# | LAK++ | HPC¶ | HPC | pre-B1-160+++ | LAK1-164++ | HPC++ |
| 104 | ND | LAK1-161++ | HPC | —1-162 | (pre-B+) | (LAK+) | HPC |
| 103 | ND | — | — | — | — | — | — |
| CD34− | |||||||
| 106 | ND | —# | —# | —# | —# | (LAK+)# | HPC |
| 105 | — | — | — | HPC# | — | — | HPC |
| 104 | ND | — | — | — | — | — | — |
| Plated Cell No./Well . | Experiment 1* . | Experiment 2† . | Experiment 3† . | Experiment 4† . | |||
|---|---|---|---|---|---|---|---|
| IL-2 + IL-7 . | IL-2 . | IL-7 . | IL-2 . | IL-7 . | IL-2 . | IL-7 . | |
| CD34+ | |||||||
| 105 | LAK‡ + 1-153/(pre-B+)1-155# | LAK++ | HPC¶ | HPC | pre-B1-160+++ | LAK1-164++ | HPC++ |
| 104 | ND | LAK1-161++ | HPC | —1-162 | (pre-B+) | (LAK+) | HPC |
| 103 | ND | — | — | — | — | — | — |
| CD34− | |||||||
| 106 | ND | —# | —# | —# | —# | (LAK+)# | HPC |
| 105 | — | — | — | HPC# | — | — | HPC |
| 104 | ND | — | — | — | — | — | — |
Different numbers of sorted CD34+ EB cells and CD34− EB cells were cultured on a confluent layer of OP9 cells established in a six-well plate. Hematopoietic progenitor cells (HPCs) proliferating under the OP9 stroma cells were monitored. When the culture reached near-saturation, cells were harvested and surface expression of B220, CD19, and CD43 was analyzed by FACScan. B220+ CD19− CD43− cytotoxic cells were designated as LAK; B220+ CD19+CD43+ small noncytotoxic cells as pre-B; and noncytotoxic cells that ceased proliferating before analysis as HPC.
Abbreviation: ND, not determined.
Continuously cultured in IL-2 (50 ng/mL) and IL-7 (5 ng/mL).
As described under Experimental Procedures, on day 7, added cytokine was changed to either IL-2 or IL-7.
Type I LAK cells (LAK34a-1) were derived from this culture.
Relative frequency of LAK, pre-B, and HPC is shown based on culture period required for the cells to proliferate to a near-saturation level. +++: the culture was saturated, and transferred to a T25 flask within 2 weeks, ++: in 3 weeks, and + in 4 weeks or longer.
LAK or pre-B in parentheses indicates that either the culture was lost mainly as a result of ES-like cell out growth or the cell failed to expand after the first round of surface marker analysis.
#ES-like cells outgrew.
¶HPC that initially grew under the OP9 layer and ceased proliferating before reaching to a near saturation level are indicated as HPC without +, ++, or +++.
EB34-pre-B cells were derived from this culture.
Type I LAK cells (LAK34a-3) were derived from this culture.
Type II LAK cells (LAK34a-2) were derived from this culture.
No sign of hematopoietic cell proliferation.