Figure 1.
Photographic examples representing the different scores of blastocysts donated for stem cell derivation.
HS475. Fully hatched blastocyst with clear tightly packed ICM. This embryo generated a new hESC line, HS475. Exp462. Hatching blastocyst with average number of cells in ICM, even trophectoderm layer. Exp472. Hatching blastocyst. Trophectoderm layer is uneven and the cells in the ICM are few in number and loosely packed. Exp404. Fully expanded blastocyst with clear tightly packed ICM and even trophectoderm layer. Exp439. Fully expanded blastocyst, with thin zona pellucida. Small ICM and trophectoderm cells of different size. Exp412. Fully developed blastocoel. Zona pellucida is still thick, meaning that the blastocyst is not fully expanded. Exp367. Early blastocyst. HS361. Early blastocyst with blastocoels filling less than 50% of embryo volume. This embryo generated the hESC line HS361. Exp457. This embryo is still compacted and has not reached blastocyst stage. All pictures were taken with a 40x objective, phase contrast or DIC, scale bar 50 µm. (Exp = experiment number. HS is used as a prefix for established hESC lines at Karolinska Institutet).
Figure 2.
The successful derivation of the hESC line HS429, derived from a 3PN embryo.
(A) The blastocyst with the score 4CB before inner cell mass isolation. (B) The mechanically isolated inner cell mass on human fibroblast feeder layer. (C) One day after isolation the inner cell mass has attached. (D) Outgrowth four days after isolation. (E) Lower magnification of the outgrowth 16 days after inner cell mass isolation, now with the typical stem cell colony morphology. From this outgrowth a small piece had been mechanically passaged four days earlier. (F) A colony of the hESC line HS429 at passage two. (Original magnification A–D 40x and E–F 400x). Normal karyotype 46,XX of the line HS429 at passage eight.
Table 1.
Data from all our 30 established hESC lines at Karolinska Institutet.
Figure 3.
Summary of blastocyst scores and efficiency in derivation.
Percentage of successful establishment of permanent hESC lines, for each criteria, is includes above the staple for established hESC lines. (A) Day of inner cell mass (ICM) isolation, number of isolations performed on embryos at different days after fertilisation, number of outgrowths from these embryos and the number of successful derivations of hESC lines. (B) Expansion Score. Number of isolations performed on embryos with different expansion scores, number of outgrowths and successful derivations. (C) ICM Score. Number of isolations performed on embryos with different ICM scores, number of outgrowths and established hESC lines from these embryos. (D) Trophectoderm Score. Number of isolations performed on embryos with different expansion scores, number of outgrowths and established hESC lines.
Table 2.
Differences of number of cells day two.