CRISPR Gene Editing in Stem Cells

These experiments are labor intensive and time consuming.

Moreover, only 25% of blastocysts derived from genetic

crosses are homozygous mutants, posing a limitation for efficient

complementation. CRISPR-Cas9 mediated zygote genome edit-

ing offers a faster and more efficient one-step process for gener-

ating mice carrying homozygous mutations, thereby providing a

robust interspecies blastocyst complementation platform. Addi-

tionally, the multiplexing capability of CRISPR-Cas9 (Cong et al.,

2013; Yang et al., 2015) could potentially be harnessed for multi-

lineage complementation. For example, in the case of the

pancreas, one might hope to eliminate both the pancreatic pa-

renchyma and vasculature of the host to generate a more com-

plete xenogeneic pancreas. Despite the advantages, there are

several technical limitations of the CRISPR-Cas9 blastocyst

complementation system that need to be overcome before un-

locking its full potential. First, gene inactivation relies on the

error-prone, non-homologous end joining (NHEJ) pathway,

which is often unpredictable. In-frame mutations and mosaicism

are among the factors that may affect outcomes. A more predict-

able targeted gene inactivation strategy that utilizes homologous

recombination (HR) is still inefficient in the zygote. Second, each

embryo must be injected twice when using this system and em-

bryosmust be cultured in vitro for several days before ET, thereby

compromising embryo quality. Technical advancements that

include a more robust gene-disruption strategy (e.g., targeted

generation of frameshift mutations via homology independent

targeted integration [Suzuki et al., 2016]), alternative CRISPR/

Cas9 delivery methods, and improved culture conditions for

manipulated embryos will likely help improve and optimize the

generation of organogenesis-disabled hosts.

We observed a slower clearance of an intraperitoneally in-

jected glucose load for

Pdx1

than

Pdx1

rat-mouse chi-

meras, while both were slower than wild-type mouse controls

(Figure S2C). While this result may seem to contradict a previ-

ous report (Kobayashi et al., 2010), the discrepancy is likely

due to the development of autoimmune type inflammation that

is often observed in adult rat-mouse (chimeras made by injec-

tion of rat PSCs into mouse blastocyst, data not shown)

(>7 months, this study) and mouse-rat chimeras (chimeras

made by injection of mouse PSCs into rat blastocyst; H. Nakau-

chi, personal communication), which is less evident in young

chimeras ( 8 weeks; Kobayashi et al. 2010). Interestingly

though, we did observe a similarly slower clearance of glucose

load in wild-type rats, although the initial spike was much lower

in rats compared to mice or chimeras (Figure S2C). Thus, the rat

cellular origin might also have played a role in the different GTT

responses observed.

Rodent ESCs/iPSCs, considered as the gold standard cells for

defining naive pluripotency, can robustly contribute to intra- and

inter-species chimeras within rodent species. These and other

results have led to the assumption that naive PSCs are the cells

of choice when attempting to generate interspecies chimeras

involving more disparate species. Here, we show that rodent

PSCs fail to contribute to chimera formation when injected into

pig blastocysts. This highlights the importance of other contrib-

uting factors underlying interspecies chimerism that may

include, but not limited to, species-specific differences in

epiblast and trophectoderm development, developmental ki-

netics, and maternal microenvironment.

To date, and taking into consideration all published studies

that have used the mouse as the host species, it is probably

appropriate to conclude that interspecies chimera formation

involving hPSCs is inefficient (De Los Angeles et al., 2015). It

has been argued that this apparent inefficiency results from spe-

cies-specific differences between human and mouse embryo-

genesis. Therefore, studies utilizing other animal hosts would

help address this important question. Here we focused on two

species, pig and cattle, from a more diverse clade of mammals

and found that naive and intermediate, but not primed, hiPSCs

could robustly incorporate into pre-implantation host ICMs.

Following ET, we observed, in general and similar to the mouse

studies, low chimera forming efficiencies for all hiPSCs tested.

Interestingly, injected hiPSCs seemed to negatively affect

normal pig development as evidenced by the high proportion

of growth retarded embryos. Nonetheless, we observed that

FAC-hiPSCs, a putative intermediate PSC type between naive

and primed pluripotent states, displayed a higher level of chime-

rism in post-implantation pig embryos. IHC analyses revealed

that FAC-hiPSCs integrated and subsequently differentiated in

host pig embryos (as shown by the expression of different line-

age markers, and the lack of expression of the pluripotency

marker OCT4). Whether the degree of chimerism conferred by

FAC-hiPSCs could be sufficient for eliciting a successful inter-

species human-pig blastocyst complementation, as demon-

strated herein between rats and mice, remains to be demon-

strated. Studies and approaches to improve the efficiency and

level of hPSC interspecies chimerism (Wu et al., 2016), such as

matching developmental timing, providing a selective advantage

for donor hPSCs, generating diverse hPSCs with a higher

chimeric potential and selecting a species evolutionarily closer

to humans, among others parameters, will be needed.

The procedures and observations reported here on the capa-

bility of human pluripotent stem cells to integrate and differentiate

in a ungulate embryo, albeit at a low level and efficiency, when

Figure 6. Chimeric Contribution of hiPSCs to Post-implantation Pig Embryos

(A) Representative bright field (left top) fluorescence (left bottom and middle) and immunofluorescence (right) images of GFP-labeled FAC-hiPSCs derivatives in a

normal size day 28 pig embryo (FAC #1). Scale bar, 100

(B) Representative immunofluorescence images showing chimeric contribution and differentiation of FAC-hiPSCs in a normal size, day 28 pig embryo (FAC #1).

FAC-hiPSC derivatives are visualized by antibodies against GFP (top), TUJ1, SMA, CK8 and HuNu (middle). (Bottom) Merged images with DAPI. Insets are higher

magnification images of boxed regions. Scale bar, 100

(C) Representative gel images showing genomic PCR analyses of pig embryos derived from blastocyst injection of 2iLD-iPSCs (surrogates #8164 and #20749)

and FAC-hiPSCs (surrogates #9159 and #18771) using a human specific Alu primer. A pig specific primer Cyt b was used for loading control. nc, negative control

with no genomic DNA loaded. pc, positive controls with human cells. Pig 1D, 1G, and 1I, pig controls. ID, surrogate and pig embryos.