Table 3.
Potency of E12.5 TEC is changed by overnight culture.
| Location and number of mGFP+ foci in recovered graft | ||||
|---|---|---|---|---|
| Graft ID | Cortex | Medulla | CMJ | Other |
| 30-40 E12.5 PLET1+mGFP+ cells injected into each E12.5 lobe and cultured O/N before grafting | ||||
| G1-30/40-O/N | 15-20 | 3 | 3 | |
| G2-30/40-O/N | 10-15 | 10-15 | 0 | |
| G3-30/40-O/N | 20-30 | 0 | 0 | |
| G4-30/40-O/N | 15-20 | 5 | 0 | |
| 30-40 E12.5 PLET1+mGFP+ cells cultured overnight then injected into uncultured E12.5 lobes and grafted within 2 hours | ||||
| G2 | 15-20 | 1-5 | 1-5 | |
| G3 | 1-5 | 1-5 | 0 | |
| G4 | 35-40 | 5-10 | 0 | |
| E12.5 lobes cultured overnight then injected with 30-40 E12.5 PLET1+mGFP+ cells and grafted within 2 hours | ||||
| G1 | 1-5 | 5-10 | 1-5 | |
| G2 | 15-20 | 1-5 | 0 | |
| G3 | 1-5 | 1-5 | 1-5 | |
| 1 E12.5 PLET1+mGFP+ cell injected into each E12.5 lobe and cultured O/N before grafting | ||||
| G1-1-ON | 3 | 1* | 0 | |
| G2-1-ON | 1-5 | 1-5 | 0 | |
| G3-1-ON | 1-5 | 1-5 | 0 | |
| G4-1-ON | 1-5 | 1-5 | 0 | |
*Very large medullary focus.
Table shows distribution of foci in grafted E12.5 thymic lobes that had been injected with the number of E12.5 mGFP+PLET1+ TEC shown. The injected lobes were cultured overnight before grafting under the kidney capsule. Grafts were recovered for analysis after two-three weeks. mGFP+ foci were scored visually for regional localization based on the DAPI, α-K14, Ly51 and CDR1 staining. 30-40 E12.5 mGFP+PLET1+ TEC injected into E12.5 lobe then cultured, n=4 independent grafts of which 4 grafts contained mGFP+ cells (n=4/4); 30-40 E12.5 mGFP+PLET1+ TEC cultured then injected into E12.5 lobe, n≥5 independent grafts of which 3 grafts contained mGFP+ cells, (n=3/≥5); E12.5 lobes cultured overnight then injected with 30-40 E12.5 mGFP+PLET1+ TEC, n=18 independent grafts of which 3 grafts contained mGFP+ cells (n=3/18); 1 E12.5 mGFP+PLET1+ TEC injected into E12.5 lobe, n=22 independent grafts of which 4 grafts contained mGFP+ cells (n=4/22). Only recovered grafts that contained mGFP+ TEC upon visual inspection were analyzed by immunohistochemistry. Contribution to the medulla appeared greater in grafts that were cultured overnight (O/N) although one graft was observed with contribution to only the cortex. N for each condition represents an independent graft; at least three biologically independent replicates were performed for each injection condition. The proportion of cTEC-fated mGFP+ cells in grafted lobes that had not been (and in which no cells had been) cultured ( Table 1 ) with that in grafted lobes that had been cultured overnight before grafting ( Table 3 ) was statistically significant as shown by a pooled two-proportion z-test, which gave a z-score of z=3.03 and ap-value of p-val=0.002.