Results of CRU assays of cultured CFSE-labeled cells
| CFSE+ Cell Phenotype Cultured . | Total Time In Vitro3-150 . | Fraction of Cells Injected . | No. of Cells/ Mouse (×103) . | No. of Positive Mice/Total No. of Mice . | Repopulation (% Donor Cells) . | CRU Frequency . | |||
|---|---|---|---|---|---|---|---|---|---|
| 3 mo . | 6 mo . | 3 mo . | 6 mo . | 3 mo . | 6 mo . | ||||
| Lin− | Overnight culture/post CFSE sort | total cells | 1.0 3.0 6.0 | 3/4 5/5 6/6 | 3/4 4/5 5/5 | 9 ± 4 27 ± 7 51 ± 2 | 24 ± 5 50 ± 7 55 ± 7 | 1/660 (1/3901/1800)3-151 | 1/1300 (1/8401/1900)3-151 |
| 3 days | undivided cells | 0.3 | 4/5 | 4/5 | 17 ± 5 | 18 ± 7 | 1/190 (1/63-550) | 1/190 (1/63-1/550) | |
| 3 days | divided cells | 1.2 3.0 7.5 | 0/12 1/6 0/2 | 0/12 1/6 0/2 | — 4 ± 4 — | — 1 ± 1 — | 1/46,000 (1/64001/330,000) | 1/46,000 (1/64001/330,000) | |
| LD | overnight culture/post CFSE sort | total cells | 9 23 50 | 6/22 5/18 8/14 | 7/22 5/17 6/13 | 10 ± 3 8 ± 3 24 ± 11 | 9 ± 3 4 ± 2 15 ± 7 | 1/52,000 (1/34,0001/83,000) | 1/54,000 (1/33,0001/88,000) |
| 4 days | undivided cells | 4.2 | 0/12 | 0/12 | — | — | <1/52,0003-152 (1/74001/370,000) | <1/52,0003-152 (1/74001/370,000) | |
| 4 days | divided cells | 10 17 25 70 | 3/16 0/5 4/11 2/2 | 3/16 1/5 5/10 1/1 | 3 ± 1 — 15 ± 6 32 ± 17 | 1 ± 0.5 2 ± 0.8 14 ± 9 20 | 1/56,000 (1/29,0001/110,000) | 1/43,000 (1/23,0001/80,000) | |
| Lin−Sca-1+ | overnight culture/post CFSE sort | total cells | 0.3 1.0 3.0 | 4/12 6/12 9/10 | 3/11 6/11 10/11 | 26 ± 8 20 ± 8 33 ± 9 | 9 ± 5 29 ± 15 66 ± 7 | 1/1,200 (1/7301/2000) | 1/1,300 (1/7601/2100) |
| 10 days | undivided cells | 0.06 | 0/10 | 0/10 | — | — | <1/6303-152 (1/90-1/4500) | <1/6303-152 (1/90-1/4500) | |
| 10 days | divided cells | 36 108 360 | 0/7 3/7 7/10 | 0/7 3/7 6/10 | — 9 ± 5 13 ± 3 | — 5 ± 3 8 ± 1 | 1/290,000 (1/150,0001/560,000) | 1/350,000 (1/180,0001/700,000) | |
| CFSE+ Cell Phenotype Cultured . | Total Time In Vitro3-150 . | Fraction of Cells Injected . | No. of Cells/ Mouse (×103) . | No. of Positive Mice/Total No. of Mice . | Repopulation (% Donor Cells) . | CRU Frequency . | |||
|---|---|---|---|---|---|---|---|---|---|
| 3 mo . | 6 mo . | 3 mo . | 6 mo . | 3 mo . | 6 mo . | ||||
| Lin− | Overnight culture/post CFSE sort | total cells | 1.0 3.0 6.0 | 3/4 5/5 6/6 | 3/4 4/5 5/5 | 9 ± 4 27 ± 7 51 ± 2 | 24 ± 5 50 ± 7 55 ± 7 | 1/660 (1/3901/1800)3-151 | 1/1300 (1/8401/1900)3-151 |
| 3 days | undivided cells | 0.3 | 4/5 | 4/5 | 17 ± 5 | 18 ± 7 | 1/190 (1/63-550) | 1/190 (1/63-1/550) | |
| 3 days | divided cells | 1.2 3.0 7.5 | 0/12 1/6 0/2 | 0/12 1/6 0/2 | — 4 ± 4 — | — 1 ± 1 — | 1/46,000 (1/64001/330,000) | 1/46,000 (1/64001/330,000) | |
| LD | overnight culture/post CFSE sort | total cells | 9 23 50 | 6/22 5/18 8/14 | 7/22 5/17 6/13 | 10 ± 3 8 ± 3 24 ± 11 | 9 ± 3 4 ± 2 15 ± 7 | 1/52,000 (1/34,0001/83,000) | 1/54,000 (1/33,0001/88,000) |
| 4 days | undivided cells | 4.2 | 0/12 | 0/12 | — | — | <1/52,0003-152 (1/74001/370,000) | <1/52,0003-152 (1/74001/370,000) | |
| 4 days | divided cells | 10 17 25 70 | 3/16 0/5 4/11 2/2 | 3/16 1/5 5/10 1/1 | 3 ± 1 — 15 ± 6 32 ± 17 | 1 ± 0.5 2 ± 0.8 14 ± 9 20 | 1/56,000 (1/29,0001/110,000) | 1/43,000 (1/23,0001/80,000) | |
| Lin−Sca-1+ | overnight culture/post CFSE sort | total cells | 0.3 1.0 3.0 | 4/12 6/12 9/10 | 3/11 6/11 10/11 | 26 ± 8 20 ± 8 33 ± 9 | 9 ± 5 29 ± 15 66 ± 7 | 1/1,200 (1/7301/2000) | 1/1,300 (1/7601/2100) |
| 10 days | undivided cells | 0.06 | 0/10 | 0/10 | — | — | <1/6303-152 (1/90-1/4500) | <1/6303-152 (1/90-1/4500) | |
| 10 days | divided cells | 36 108 360 | 0/7 3/7 7/10 | 0/7 3/7 6/10 | — 9 ± 5 13 ± 3 | — 5 ± 3 8 ± 1 | 1/290,000 (1/150,0001/560,000) | 1/350,000 (1/180,0001/700,000) | |
Data are pooled from like experiments (see Table 1). In each experiment, essentially all cells harvested from the culture were used for CRU assays. Repopulation values shown are the mean ± SEM (standard error of the mean).
Total time in vitro includes the initial day of culture involved in the CFSE labeling procedure prior to isolating the input CFSE+ cells.
Numbers in parentheses represent upper and lower limits of the 95% confidence interval.
The value shown is an estimate of the upper limit of CRU frequency calculated by assuming that one of the recipients had been positive.