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simplified version of NHSM, have a significant potential for germ
cell induction, a distinguishing feature between naive mESCs
and primed mEpiSCs (Irie et al., 2015). Thus, we also culture-
adapted NHSM-hiPSCs in 4i medium (4i-hiPSCs), resulting in
stable 4i-hiPSCs with similar morphological and molecular char-
acteristics to parental NHSM-hiPSCs (Figure 4B). In addition, we
generated another type of hiPSC by direct reprogramming of
HFFs in a modified mEpiSC medium containing bFGF, Activin-
A, and CHIR99021 (FAC; Figure 4A). mEpiSCs cultured in FAC
medium exhibited features characteristic of both naive mESCs
and primed mEpiSCs, supporting an intermediate pluripotent
state (Tsukiyama and Ohinata, 2014). hiPSCs generated and
cultured in FAC medium (FAC-hiPSCs) displayed a colony
morphology intermediate between that of 2iLD- and primed
hiPSCs, with less defined borders (Figure 4B). 2iLD-hiPSCs,
NHSM-hiPSCs, 4i-hiPSCs, and FAC-hiPSCs could all be stably
maintained long term in culture, preserving normal karyotypes
and the homogeneous, nuclear localization of OCT4 protein
(Figure 4B; data not shown). Notably, similar to hiPSCs grown
in naive cultures (2iLD-hiPSCs, NHSM-hiPSCs, 4i-hiPSCs),
FAC-hiPSCs could also be efficiently propagated by single-cell
dissociation without using a ROCK kinase inhibitor. After inject-
ing cells into the kidney capsule of immunodeficient NSG mice,
all of these hiPSCs formed teratomas that consisted of tissues
from all three germ layers: endoderm, mesoderm, and ectoderm
(Figure S3A). To facilitate the identification of human cells in sub-
sequent chimera experiments, we labeled hiPSCs with either
green fluorescence protein (GFP) or hKO fluorescence markers.
Chimeric Contribution of hiPSCs to Pig and Cattle
Blastocysts
The ability to integrate into the inner cell mass (ICM) of a blasto-
cyst is informative for evaluating whether hiPSCs are compatible
with pre-implantation epiblasts of the ungulate species. This is
also one of the earliest indicators of chimeric capability. We
therefore evaluated interspecies chimeric ICM formation by in-
jecting hiPSCs into blastocysts from two ungulate species, pig
and cattle.
Cattle-assisted reproductive technologies, such as in vitro
embryo production, are well established given the commercial
benefits of improving the genetics of these animals. Cattle also
serve as a research model because of several similarities to hu-
man pre-implantation development (Hansen, 2014; Hasler,
2014). Using techniques for producing cattle embryos in vitro,
we developed a system for testing the ability and efficiency of
hiPSCs to survive in the blastocyst environment and to integrate
into the cattle ICM (Figure 4C). Cattle embryos were obtained by
in vitro fertilization (IVF) using in vitro matured oocytes collected
from ovaries obtained from a local slaughterhouse. The tightly
connected cells of the blastocyst trophectoderm from large live-
stock species, such as pig and cattle, form a barrier that compli-
cates cell microinjection into the blastocoel. Thus, microinjection
often results in embryo collapse and the inability to deposit the
cells into the embryo. To facilitate cell injection we employed a
laser-assisted approach, using the laser to perforate the zona
pellucida and to induce damage to a limited number of trophec-
toderm cells. This allowed for easy access into the blastocyst
cavity for transferring the human cells (Figure S3B). Furthermore,
the zona ablation and trophectoderm access allowed use a
blunt-end pipette for cell transfer, thus minimizing further em-
bryo damage. This method resulted in a nearly 100% injection
effectiveness and >90% embryo survival.
To determine whether hiPSCs could engraft into the cattle
ICM, we injected ten cells from each condition into cattle blasto-
cysts collected 7 days after fertilization. After injection, we
cultured these blastocysts for additional 2 days before analysis.
We used several criteria to evaluate the chimeric contribution of
hiPSCs to cattle blastocysts: (1) average number of human cells
in each blastocyst, (2) average number of human cells in each
ICM, (3) percentage of blastocysts with the presence of human
cells in the ICM, (4) percentage of SOX2+ human cells in the
ICM, and (5) percentage of human cells in the ICM that are
SOX2+ (Figure 4C). Our results indicated that both naive and in-
termediate (but not primed) hiPSCs could survive and integrate
into cattle ICMs, albeit with variable efficiencies (Figures 4D
and S3C–S3E; Table S4). Compared with other cell types,
4i-hiPSCs exhibited the best survival (22/23 blastocysts con-
tained human cells), but the majority of these cells lost SOX2
expression (only 13.6% of human cells remained SOX2+). On
average, 3.64 4i-hiPSCs were incorporated into the ICM.
NHSM-hiPSCs were detected in 46 of 59 injected blastocysts,
with 14.41 cells per ICM. Of these, 89.7% remained SOX2+.
For 2iLD-hiPSCs, 40 of 52 injected blastocysts contained human
cells, with 5.11 cells per ICM, and 69.9% of the ICM-incorpo-
rated human cells remained SOX2+. FAC-hiPSCs exhibited
moderate survival rate (65/101) and ICM incorporation efficiency
(39/101), with an average of 2.31 cells incorporated into the ICM,
and 89.3% remaining SOX2+.
We also performed ICM incorporation assays by injecting
hiPSCs into pig blastocysts. Because certain complications
are frequently associated with pig IVF (Abeydeera, 2002;
Grupen, 2014) (e.g., high levels of polyspermic fertilization), we
used a parthenogenetic activation model, which enabled us to
efficiently produce embryos that developed into blastocysts
(King et al., 2002). Pig oocytes were obtained from ovaries
collected at a local slaughterhouse. Once the oocytes were
matured in vitro, we removed the cumulus cells and artificially
activated the oocytes using electrical stimulation. They were
then cultured to blastocyst stage (Figure 4C). We injected ten
hiPSCs into each pig parthenogenetic blastocyst and evaluated
their chimeric contribution after 2 days of in vitro culture (Figures
4C and S3C–S3E; Table S4). Similar to the results in cattle, we
found that hiPSCs cultured in 4i and NHSM media survived bet-
ter and yielded a higher percentage of blastocysts harboring
human cells (28/35 and 37/44, respectively). Also, among all
blastocysts containing human cells, we observed an average
of 9.5 cells per blastocyst for 4i-hiPSCs and 9.97 cells for
NHSM-hiPSCs. For NHSM-hiPSCs, 19/44 blastocysts had
human cells incorporated into the ICM. In contrast, only 6/35
blastocysts had 4i-hiPSCs localized to the ICM. For 2iLD-
hiPSCs, we observed an average of 5.7 cells per blastocyst,
with 2.25 human cells localized to the ICM. For FAC-hiPSCs,
an average of 3.96 and 1.62 human cells were found in the blas-
tocyst and ICM, respectively. Once incorporated into the ICM,
82.2%, 72%, 60.9%, and 40% of 2iLD-, 4i-, NHSM-, and FAC-
hiPSCs, respectively, stained positive for the pluripotency
Cell
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