CRISPR Gene Editing in Stem Cells

Figure 4. Generation and Interspecies ICM

Incorporation of Different Types of hiPSCs

(A) Schematic of the strategy for generating naive,

intermediate, and primed hiPSCs.

(B) (Top) Representative bright-field images

showing the colony morphologies of naive (2iLD-,

4i-, and NHSM-hiPSCs) and intermediate (FAC-

hiPSCs) hiPSCs. Bottom, representative immu-

nofluorescence images of naive and intermediate

hiPSCs stained with an anti-OCT4 antibody. Red,

OCT4; blue, DAPI. Scale bar, 100

producing cattle and pig blastocysts obtained

from in vitro fertilization (IVF) and parthenoactiva-

tion, respectively. Blastocysts were subsequently

used for laser-assisted blastocyst injection of

hiPSCs. After hiPSC injection, blastocysts were

cultured in vitro for 2 days before fixation and

analyzed by immunostaining with an anti-HuNu

and an anti-SOX2 antibodies. Criteria to evaluate

the survival of human cells, as well as the degree

and efficiency of ICM incorporation are shown in

the blue box.

(D) Number of hiPSCs that integrated into the

cattle (left) and pig (right) ICMs after ten hiPSCs

were injected into the blastocyst followed by

2 days of in vitro culture. Red line, the average

number of ICM-incorporated hiPSCs. Blue dot,

the number of ICM-incorporated hiPSCs in each

blastocyst.