Table 2:

Predicted number of well-formed structures based on observed experimental efficiencies.

	EiTiX embryo			ETiX embryo
Day of experiment	Predicted number of structures	Average experimental efficiency of progression to the next day (n= 3–5 experiments)	Standard deviation of experimental efficiency	Predicted number of structures	Average experimental efficiency of progression to the next day (n= 17 experiments)	Standard deviation of experimental efficiency
0	1200 (per AggreWell)	/	/	1200 (per AggreWell)	/	/
4	186	15.5%	0.68	261	21.7%	1.33
5	45	24.4%	0.85	54	21.0%	8.50
6	29	65.4%	14.8	40	74.7%	23.2
7	20	72.1%	13.2	30	74.6%	19.2
8	15	75.0%	16.7	22	72.1%	22.8

Day 4 efficiency was determined by immunofluorescence analysis of all structures collected from one AggreWell (typically around 700–1000 structures, with some loss of structures during the washing steps in immunofluorescence). Correct Day 4 structures display the expected spatial expression of canonical lineage markers as shown in Figure 5.

Day 5, 6, 7 and 8 efficiencies were calculated by the percentage of successful structures out of those selected on the previous day. Note that on Day 4, 35–55 structures were selected from each AggreWell set up on Day 0 (see Step 49), while on Day 5, 10–15 structures obtained from 6 starting AggreWells were selected for further culture and the subsequent experiment efficiencies (see Step 58).