Table 1. Quantitative comparison of pluripotency and differentiation phenotypes.
Proliferation was measured by seeding 4 96well plate wells, imaging whole wells at day 0, 1, 3, and 6, and scoring images for total cells (DAPI+) and subtracting the number of Sytox orange+ (dead) cells = total live cells. Total growth: table shows mean cells/line/genotype/day and standard deviation; n = 2 and n = 3 lines per WT or KO genotype respectively. Total cells is the sum of cells in four replicate wells/line. Normalized growth: Each line was normalized to its own day 0 value. Curve Fit Parameters: each line was fit with an exponential growth curve and the mean growth constants were computed and found not significantly different. Colony Size: whole colonies were segmented using MultiWavelength Cell Scoring adjusting the size and intensity thresholds so that entire colonies were segmented by DAPI signal, rather than individual cells. Mean values for n colonies counted for each line at day 0 and day six are shown, unit is μm2. No significant differences were found between genotype at either day. Proliferation: Three randomly chosen 10x fields (mean 1473 cells/field) per line were scored for total DAPI +cells and cells positive for phosphohistone H3 (PH3) in Metamorph. No significant difference between genotypes was observed. No cells were positive for activated caspase-3 or showed pyknotic nuclei, indicating negligible levels of cell death across genotypes. All statistics Student’s t-test, 1 tail, 2 sample unequal variance.
| Total growth | ||||
|---|---|---|---|---|
| WT (n = 2) | SD | KO (n = 3) | SD | |
| Day 0 | 402.50 | 127.01 | 721.42 | 115.63 |
| Day 1 | 342.75 | 61.23 | 849.33 | 64.51 |
| Day 3 | 2289.50 | 334.41 | 2043.17 | 346.46 |
| Day 6 | 7919.38 | 1728.79 | 5234.17 | 1350.08 |
| Normalized growth | ||||
| WT (n = 2) | SD | KO (n = 3) | SD | |
| Day 0 | 1.000 | 0.356 | 1.000 | 0.133 |
| Day 1 | 0.864 | 0.206 | 1.388 | 0.077 |
| Day 3 | 5.689 | 1.076 | 3.518 | 0.427 |
| Day 6 | 19.802 | 5.096 | 8.124 | 1.708 |
| Curve Fit Parameters | ||||
| Growth Constant | Goodness of Fit | |||
| HB9 (wt) | 0.443 | 0.985 | ||
| WT13 | 0.489 | 0.996 | ||
| KO5 | 0.377 | 0.993 | ||
| KO6 | 0.287 | 0.994 | ||
| KO8 | 0.316 | 0.971 | ||
| Mean Growth Constant | SD | |||
| WT | 0.326 | 0.033 | ||
| KO | 0.466 | 0.046 | ||
| p-value | 0.316 | |||
| Colony size | ||||
| Day 0 | Day 6 | |||
| Mean | SD | Mean | SD | |
| HB9 (wt) (day 0, 6: n = 122, 30) | 45666.2 | 81641.1 | 133231.9 | 66024.3 |
| WT13 (day 0, 6: n = 74, 28) | 78107.7 | 94966.6 | 156237.9 | 90772.1 |
| KO5 (day 0, 6: n = 128, 24) | 40269.1 | 74822.2 | 129713.2 | 81019.6 |
| KO6 (day 0, 6: n = 69, 17) | 101383.7 | 120527.3 | 95029.8 | 42050.6 |
| KO8 (day 0, 6: n = 71, 19) | 84821.1 | 108565.5 | 145450.6 | 107648.8 |
| Day 0 | Day 6 | |||
| Mean | SD | Mean | SD | |
| WT | 61886.9 | 22939.7 | 144734.9 | 16267.7 |
| KO | 75491.3 | 31607.4 | 123397.8 | 25796.9 |
| p value | 0.618 | 0.170 | ||
| Proliferation | ||||
| % PH3+ of total DAPI | mean% PH3+ | SD | ||
| HB9 (wt) | 3.487 | WT | 3.224 | 0.372 |
| WT13 | 2.961 | PZ1KO | 3.424 | 0.218 |
| KO5 | 3.331 | T-test p value | 0.292 | |
| KO6 | 3.268 | |||
| KO8 | 3.673 | |||