Biology

Article

Interspecies Chimerism with Mammalian Pluripotent

Stem Cells

Graphical Abstract

Highlights

d Naive rat PSCs robustly contribute to live rat-mouse

chimeras

d A versatile CRISPR-Cas9 mediated interspecies blastocyst

complementation system

d Naive rodent PSCs show no chimeric contribution to post-

implantation pig embryos

d Chimerism is observed with some human iPSCs in post-

implantation pig embryos

Wu et al., 2017, Cell 168, 473–486 January 26, 2017 ª 2017 Elsevier Inc. http://dx.doi.org/10.1016/j.cell.2016.12.036

Authors

Jun Wu, Aida Platero-Luengo,

Masahiro Sakurai, ..., Emilio A. Martinez,

Pablo Juan Ross,

Juan Carlos Izpisua Belmonte

Correspondence [email protected]

In Brief

Human pluripotent stem cells robustly

engraft into both cattle and pig pre-

implantation blastocysts, but show

limited chimeric contribution to post-

implantation pig embryos.

Article

Interspecies Chimerism with Mammalian Pluripotent Stem Cells Jun Wu,1 Aida Platero-Luengo,1 Masahiro Sakurai,1 Atsushi Sugawara,1 Maria Antonia Gil,2 Takayoshi Yamauchi,1

Keiichiro Suzuki,1 Yanina Soledad Bogliotti,3 Cristina Cuello,2 Mariana Morales Valencia,1 Daiji Okumura,1,7

Jingping Luo,1 Marcela Vilariño,3 Inmaculada Parrilla,2 Delia Alba Soto,3 Cristina A. Martinez,2 Tomoaki Hishida,1

Sonia Sánchez-Bautista,4 M. Llanos Martinez-Martinez,4 Huili Wang,3 Alicia Nohalez,2 Emi Aizawa,1

Paloma Martinez-Redondo,1 Alejandro Ocampo,1 Pradeep Reddy,1 Jordi Roca,2 Elizabeth A. Maga,3

Concepcion Rodriguez Esteban,1 W. Travis Berggren,1 Estrella Nuñez Delicado,4 Jeronimo Lajara,4 Isabel Guillen,5

Pedro Guillen,4,5 Josep M. Campistol,6 Emilio A. Martinez,2 Pablo Juan Ross,3 and Juan Carlos Izpisua Belmonte1,8,* 1Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA 2Department of Animal Medicine and Surgery, University of Murcia Campus de Espinardo, 30100 Murcia, Spain 3Department of Animal Science, University of California Davis, One Shields Avenue, Davis, CA 95616, USA 4Universidad Católica San Antonio de Murcia (UCAM) Campus de los Jerónimos, N� 135 Guadalupe 30107 Murcia, Spain 5Clinica Centro Fundación Pedro Guillén, Clı́nica CEMTRO, Avenida Ventisquero de la Condesa 42, 28035 Madrid, Spain 6Hospital Clı́nico de Barcelona-IDIBAPS, Universitat de Barcelona, 08007 Barcelona, Spain 7Present address: Graduate School of Agriculture, Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi,

Nara 631-8505, Japan 8Lead Contact

*Correspondence: [email protected]

http://dx.doi.org/10.1016/j.cell.2016.12.036

SUMMARY

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a ver- satile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis- disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, inter- species blastocyst complementation might allow hu- man organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent spe- cies remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the un- gulates. We find that naı̈ve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type ex- hibits higher degree of chimerism and is able to generate differentiated progenies in post-implanta- tion pig embryos.

INTRODUCTION

Embryonic pluripotency has been captured in vitro at a spectrum

of different states, ranging from the naive state, which reflects

unbiased developmental potential, to the primed state, in which

cells are poised for lineage differentiation (Weinberger et al.,

2016; Wu and Izpisua Belmonte, 2016). When attempting to

introduce cultured pluripotent stem cells (PSCs) into a devel-

oping embryo of the same species, recent studies demonstrated

that matching developmental timing is critical for successful

chimera formation. For example, naive mouse embryonic stem

cells (mESCs) contribute to chimera formation when injected

into a blastocyst, whereas primed mouse epiblast stem cells

(mEpiSCs) efficiently engraft into mouse gastrula-stage em-

bryos, but not vice versa (Huang et al., 2012; Wu et al., 2015).

Live rodent interspecies chimeras have also been generated us-

ing naive PSCs (Isotani et al., 2011; Kobayashi et al., 2010; Xiang

et al., 2008). However, it remains unclear whether naive PSCs

can be used to generate chimeras between more distantly

related species.

The successful derivation of human PSCs (hPSCs), including

ESCs from pre-implantation human embryos (Reubinoff et al.,

2000; Thomson et al., 1998), as well as the generation of induced

pluripotent stem cells (iPSCs) from somatic cells through cellular

reprograming (Takahashi et al., 2007; Park et al., 2008; Wernig et

al., 2007; Yu et al., 2007; Aasen et al., 2008), has revolutionized

the way we study human development and is heralding a new

age of regenerative medicine. Several lines of evidence indicate

that conventional hPSCs are in the primed pluripotent state,

similar to mEpiSCs (Tesar et al., 2007; Wu et al., 2015). A number

of recent studies have also reported the generation of putative

naive hPSCs that molecularly resemble mESCs (Gafni et al.,

2013; Takashima et al., 2014; Theunissen et al., 2014). These

naive hPSCs have already provided practical and experimental

advantages, including high single-cell cloning efficiency and

facile genome editing (Gafni et al., 2013). Despite these

advances, it remains unclear how the putative higher develop-

mental potential of naive hPSCs can be used to better

Cell 168, 473–486, January 26, 2017 ª 2017 Elsevier Inc. 473

A B

C D

E F

Figure 1. Interspecies Rat-Mouse Chimeras

Derived from Rat PSCs

(A) Rat-mouse chimeras generated by rat ESCs

(DAC2). Left, an E18.5 rat-mouse chimeric fetus. Red,

hKO-labeled rat cells. Right, a 12-month-old (top) and

24-month-old (bottom) rat-mouse chimera.

(B) Chimera forming efficiencies with rat ESC lines

(DAC2 and DAC8) and rat iPSC lines (SDFE and

SDFF). n, number of embryo transfers.

(C) Representative fluorescence images showing

hKO-labeled rat ESCs (DAC2) contributed to

different tissues in the 24-month-old rat-mouse

chimera. Red, hKO-labeled rat cells. Blue, DAPI.

Scale bar, 100 mm.

(D) Representative immunofluorescence images

showing the expression of aging-related histone

marks, including H3K9me3 and H4K20me3, in the

kidney tissue of neonatal and 24-month-old chi-

meras. Scale bar, 10 mm.

(E) Levels of chimerism of rat ESCs (DAC2) in

different tissues of the 24-month-old rat-mouse

chimera. Error bars indicate SD.

(F) Rat iPSCs (SDFE) contributed to the neonatal

mouse gall bladder. Left, bright-field (top) and

fluorescence (bottom) images showing a neonatal

mouse gallbladder contained cells derived from rat

iPSCs. White arrowheads indicate the gallbladder.

Right, representative immunofluorescence images

showing the expression of a gallbladder epithelium

marker (EpCAM) by rat cells. Red, hKO-labeled rat

cells; blue, DAPI. Scale bar, 50 mm.

See also Figure S1 and Table S2.

understand human embryogenesis and to develop regenerative

therapies for treating patients.

Like naive rodent PSCs, naive hPSCs can potentially be used to

generate interspecies chimeras for studying human development

and disease, and producing functional human tissues via interspe-

cies blastocyst complementation. To date, however, all reported

attempts on generating hPSC-derived interspecies chimeras

have used the mouse as the host animal, and the results obtained

suggest that this process is rather inefficient (Gafni et al., 2013;

Theunissen et al., 2014, 2016). Although the mouse is one of the

most important experimental models for stem cell research, there

are considerable differences between humans and mice (e.g., early

post-implantation development, embryo size, gestational length,

and developmental speed), which may hinder not only the effi-

ciency but also the usefulness of human-mouse chimeric studies.

Thus, expanding the repertoire of host species may complement

this incipient but promising area of research in the field of regener-

ative medicine. In particular, interspecies chimera research of

474 Cell 168, 473–486, January 26, 2017

hPSCs using ungulates, e.g., pigs, cattle,

and sheep, could lead to improved

research models, as well as novel in vivo

strategies for (1) generating human organs

and tissues, (2) designing new drug

screening methodologies, and (3) devel-

oping new human disease models (Wu

and Izpisua Belmonte, 2015). Experiments

to empirically test and evaluate the

chimeric contribution of various types of hPSCs in the ungulates

are thus imperative, but currently lacking. To start filling this void,

we tested different types of hPSCs for their chimeric contribution

potential in two ungulate species, pigs and cattle.

RESULTS

Naive Rat PSCs Robustly Contribute to Rat-Mouse Interspecies Chimera Formation We first used rodent models to gain a better understanding of the

factors and caveats underlying interspecies chimerism with

PSCs. To this end, we used two chimeric-competent rat ESC

lines, DAC2 and DAC8 (Li et al., 2008). We labeled both lines

with a fluorescent marker, humanized kusabira orange (hKO),

for cell tracking and injected them into mouse blastocysts.

Following embryo transfer (ET) into surrogate mouse mothers,

both DAC2 and DAC8 lines gave rise to live rat-mouse chimeras

(Figures 1A and S1A). Many of the chimeras developed into

adulthood, and one chimera reached 2 years of age (Figure 1A),

indicating that the xenogeneic rat cells sustained the physiolog-

ical requirements of the mouse host without compromising its life

span. We also generated two rat iPSC lines (SDFE and SDFF)

from tail tip fibroblasts (TTFs) isolated from a neonatal

Sprague-Dawley rat and used them to generate rat-mouse chi-

meras. Similar to rat ESCs, rat iPSCs could also robustly

contribute to chimera formation in mice (Figure S1B). Overall,

the chimera forming efficiencies of all rat PSC lines tested

were �20%, consistent with a previous report (Figure 1B) (Ko- bayashi et al., 2010).

We observed contribution of rat cells to a wide range of tissues

and organs in both neonatal and aged rat-mouse chimeras (Fig-

ures 1C, S1A, and S1B). We examined aging-related histone

marks in both neonatal and aged chimeras and found that the

2-year-old chimera exhibited histone signatures characteristic

of aging (Figure 1D). We quantified the degree of chimerism in

different organs of the aged chimera via quantitative qPCR anal-

ysis of genomic DNA using a rat-specific primer (Table S2). We

found that different tissues contained different percentages of

rat cells, with the highest contribution observed in the heart

(�10%) (Figure 1E). One anatomical difference between mice and rats is that rats

lack a gallbladder. In agreement with a previous report (Kobaya-

shi et al., 2010), we also observed the presence of gallbladders in

rat-mouse chimeras (chimeras derived from injecting rat PSCs

into a mouse blastocyst). Interestingly, rat cells contributed to

the chimeric gallbladder and expressed the gallbladder epithe-

lium marker EpCAM (Figures 1F and S1C), which suggests that

the mouse embryonic microenvironment was able to unlock a

gallbladder developmental program in rat PSCs that is normally

suppressed during rat development.

A Versatile CRISPR-Cas9-Mediated Interspecies Blastocyst Complementation System Chimeric contribution of PSCs is random and varies among

different host blastocysts and donor cell lines used. To selec-

tively enrich chimerism in a specific organ, a strategy called

blastocyst complementation has been developed where the

host blastocysts are obtained from a mutant mouse strain in

which a gene critical for the development of a particular lineage

is disabled (Chen et al., 1993; Kobayashi et al., 2010; Wu and

Izpisua Belmonte, 2015). Mutant blastocysts used for comple-

mentation experiments were previously obtained from existing

lines of knockout mice, which were generated by gene targeting

in germ-line-competent mouse ESCs—a time-consuming pro-

cess. To relieve the dependence on existing mutant strains,

we developed a blastocyst complementation platform based

on targeted genome editing in zygotes. We chose to use the

CRISPR-Cas9 system, which has been harnessed for the effi-

cient generation of knockout mouse models (Wang et al.,

2013) (Figure 2A).

For proof-of-concept, we knocked out the Pdx1 gene in

mouse by co-injecting Cas9 mRNA and Pdx1 single-guide

RNA (sgRNA) into mouse zygotes. During mouse development,

Pdx1 expression is restricted to the developing pancreatic

anlagen and is a key player in pancreatic development. Mice ho-

mozygous for a targeted mutation in Pdx1 lack a pancreas and

die within a few days after birth (Jonsson et al., 1994; Offield

et al., 1996). Similarly, Pdx1�/� mice generated by the zygotic co-injection of Cas9 mRNA and Pdx1 sgRNA were apancreatic,

whereas other internal organs appeared normal (Figure S2A).

These mice survived only a few days after birth. We observed

the efficiency for obtaining Pdx1�/� mouse via CRISPR-Cas9 zygote genome editing was �60% (Figure S2F). Next, we com- bined zygotic co-injection of Cas9/sgRNA with blastocyst injec-

tion of rat PSCs, and found that rat PSC-derivatives were

enriched in the neonatal pancreas of Pdx1�/� mice and ex- pressed a-AMYLAYSE, a pancreatic enzyme that helps digest

carbohydrates (Figures 2B and S2B). Of note is that in these an-

imals the pancreatic endothelial cells were still mostly of mouse

origin, as revealed by staining with an anti-CD31 antibody (Fig-

ure 2B). Importantly, pancreas enriched with rat cells supported

the successful development of Pdx1�/� mouse host into adult- hood (>7 months), and maintained normal serum glucose levels

in response to glucose loading, as determined using the glucose

tolerance test (GTT) (Figure S2C).

Taking advantage of the flexibility of the CRISPR-Cas9 zy-

gotic genome editing, we next sought to enrich xenogenic rat

cells toward other lineages. Nkx2.5 plays a critical role in early

stages of cardiogenesis, and its deficiency leads to severe

growth retardation with abnormal cardiac looping morphogen-

esis, an important process that leads to chamber and valve for-

mation (Lyons et al., 1995; Tanaka et al., 1999). Mice lacking

Nkx2.5 typically die around E10.5 (Lyons et al., 1995; Tanaka

et al., 1999). Consistent with previous observations, CRISPR-

Cas9 mediated inactivation of Nkx2.5 resulted in marked

growth-retardation and severe malformation of the heart at

E10.5 (Figure S2D). In contrast, when complemented with rat

PSCs, the resultant Nkx2.5�/� mouse hearts were enriched with rat cells and displayed a normal morphology, and the em-

bryo size was restored to normal (Figures 2C and S2D). Of note

is that although rat PSCs rescued embryo growth and cardiac

formation in E10.5 Nkx2.5�/� mouse embryos, to date we still have not obtained a live rescued chimera (n = 12, where n is

the number of ETs). Pax6 is a transcription factor that plays

key roles in development of the eye, nose and brain. Mice ho-

mozygous for a Pax6 loss-of-function mutation lack eyes, nasal

cavities, and olfactory bulbs, and exhibit abnormal cortical

plate formation, among other phenotypes (Gehring and Ikeo,

1999). Pax6 is best known for its conserved function in eye

development across all species examined (Gehring and Ikeo,

1999). In agreement with the published work, CRISPR-Cas9

mediated Pax6 inactivation disrupted eye formation in the

E15.5 mouse embryo (Figure S2E). When complemented with

rat PSCs, we observed the formation of chimeric eyes enriched

with rat cells in Pax6�/� mouse neonate (Figures 2D and S2E). Similar to Pdx1�/�, we observed efficient generation of homo- zygous Nkx2.5�/� and Pax6�/� mouse embryos via zygotic co-injection of Cas9 mRNA and sgRNAs (Figure S2F). All DNA

sequencing results of CRISPR-Cas9 mediated gene knockouts

and gRNA sequences are summarized in Tables S1 and S2,

respectively.

In sum, for the pancreas, heart, and eye, as well as several

other organs (data not shown), we successfully generated chi-

merized organs that were enriched with rat cells, demonstrating

Cell 168, 473–486, January 26, 2017 475

A

B

C D

Figure 2. Interspecies Blastocyst Complementation via CRISPR-Cas9-Mediated Zygote Genome Editing

(A) Schematic of the CRISPR-Cas9 mediated rat-mouse blastocyst complementation strategy.

(B) Left, bright-field (top) and fluorescence (bottom) images showing the enrichment of rat cells in the pancreas of an E18.5 Pdx1�/� mouse. Li, liver; St, stomach; Sp, spleen. Yellow-dotted line encircles the pancreas. Red, hKO-labeled rat cells. Middle and right (top), representative immunofluorescence images showing rat

cells expressed a-amylase in the Pdx1�/� mouse pancreas. Blue, DAPI. Right (bottom), a representative immunofluorescence image showing that some pancreatic endothelial cells, as marked by a CD31 antibody, were not derived from rat PSCs. Scale bar, 100 mm.

(C) Bright field (left) and fluorescence (right) images showing the enrichment of rat cells in the heart of an E10.5 Nkx2.5�/� mouse. Red, hKO-labeled rat cells. (D) Bright field (top) and fluorescence (bottom) images showing the enrichment of rat cells in the eye of a neonatal Pax6�/� mouse. Red, hKO-labeled rat cells. WT, mouse control; WT+rPSCs, control rat-mouse chimera without Cas9/sgRNA injection.

See also Figure S2 and Tables S1 and S2.

476 Cell 168, 473–486, January 26, 2017

A

B C

Figure 3. Naive Rodent PSCs Fail to Contribute to Chimera Formation in Pigs

(A) Schematic of the generation and analyses of post-implantation pig embryos derived from blastocyst injection of naive rodent PSCs.

(B) Summary of the pig embryos recovered between day 21–28 of pregnancy.

(C) Genomic PCR analyses of pig embryos derived from blastocyst injection of mouse iPSCs or rat ESCs. Mouse- and rat- specific mtDNA primers were used for

the detection of chimeric contribution from mouse iPSCs and rat ESCs, respectively. Pig-specific mtDNA primers were used for the control.

See also Tables S2 and S3.

the efficacy and versatility of the CRISPR-Cas9 mediated inter-

species blastocyst complementation platform.

Naive Rodent PSCs Do Not Contribute to Chimera Formation in Pigs It is commonly accepted that the key functional feature of naive

PSCs is their ability to generate intraspecies germline chimeras

(Nichols and Smith, 2009). Studies in rodents also support the

notion that attaining the naive pluripotent state is the key step

in enabling chimera formation across species boundaries (Xiang

et al., 2008; Isotani et al., 2011; Kobayashi et al., 2010). However,

it has not yet been tested whether naive rodent PSCs can

contribute to chimera formation when using a non-rodent host.

To further examine the relationship between naive PSCs and

interspecies chimerism, we injected rat ESCs into pig blasto-

cysts followed by ET to recipient sows. In addition to rat ESCs,

we also used a germline competent mouse iPSC line (Okita

et al., 2007). Several criteria were used to determine the chimeric

contribution of rodent cells in pig embryos, namely, (1) detection

of fluorescence (hKO) signal, (2) immunohistochemical (IHC) la-

beling of embryo sections with an anti-hKO antibody, and (3)

genomic PCR with mouse- or rat-specific primers targeting mito-

chondrial DNA (mtDNA) (Figure 3A). We terminated the preg-

nancy between day 21–28 of pig development and collected

embryos derived from the injection of mouse iPSCs or rat

ESCs into pig blastocyst (26 and 19 embryos, respectively) (Fig-

ure 3B; Table S3). We failed to detect any hKO signal in both

normal size and growth retarded embryos (Figure 3B). We next

sectioned the pig embryos and stained them with an antibody

against hKO. Similarly, we did not detect any hKO-positive cells

in the embryonic sections examined (data not shown). Finally, we

employed a more sensitive test, using genomic PCR to amplify

rat- or mouse-specific mtDNA sequences (pig-specific mtDNA

primers served as the loading control) (Table S2). Consistently,

genomic PCR analyses did not detect any rodent contribution

to the pig embryos (Figure 3C). Taken together, although naive

rodent PSCs can robustly contribute to rodent-specific interspe-

cies chimeras, our results show that these cells are incapable of

contributing to normal embryonic development in pigs.

Generation of Naive, Intermediate, and Primed hiPSCs Next, we sought to systematically evaluate the chimeric compe-

tency of hPSCs in ungulate embryos. We generated hiPSCs

using several reported naive PSC culture methods, a culture

protocol supporting a putative intermediate pluripotent state

between naive mESCs and primed mEpiSCs (Tsukiyama and

Ohinata, 2014), and a primed culture condition (Figure 4A).

Mouse ground state culture condition (2iL) induces the differen-

tiation of primed hPSCs. However, when combined with the

forced expression of NANOG and KLF2 (NK2), transcription fac-

tors that help to maintain murine naive pluripotency, 2iL culture

can stabilize hPSCs in an immature state (Takashima et al.,

2014; Theunissen et al., 2014). We generated doxycycline

(DOX)-inducible NK2-expressing naive hiPSCs cultured in 2iL

medium from primed hiPSCs (2iLD-hiPSCs). Transgene-free

primed hiPSCs were reprogramed from human foreskin fibro-

blasts (HFFs) using episomal vectors (Okita et al., 2011). For

comparison, we also generated naive hiPSCs from HFFs

using the NHSM culture condition (Gafni et al., 2013) (NHSM-

hiPSCs). It has been shown that cells grown in 4i medium, a

Cell 168, 473–486, January 26, 2017 477

A

B

C

D

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 mm.

(C) Schematic of the experimental procedures for

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.

See also Figure S3 and Table S4.

478 Cell 168, 473–486, January 26, 2017

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 168, 473–486, January 26, 2017 479

marker SOX2. These results indicate that both naive and inter-

mediate hiPSCs seem to perform better when injected into cattle

than pig blastocysts. This suggests a different in vivo blastocyst

environment in pig and cattle, with the cattle blastocysts

providing an environment that is more permissive for hiPSC inte-

gration and survival.

Chimeric Contribution of hiPSCs to Post-implantation Pig Embryos Although ICM incorporation of hiPSCs is the necessary first step

to contribute to the embryo proper of host animals, it has limited

predictive value for post-implantation chimera formation, as

other factors are involved. Next, we investigated if any of the

naive and intermediate hiPSCs that we generated, which

showed robust ICM incorporation in pre-implantation blasto-

cysts, could contribute to post-implantation development

following ET. The pig has certain advantages over cattle for ex-

periments involving post-implantation embryos, as they are a

polytocus species, and are commonly used as a translational

model given their similarities to humans concerning organ phys-

iology, size, and anatomy. We thus chose the pig for these ex-

periments. Since there was little to no contribution of primed

hiPSCs, even at the pre-implantation blastocyst stage, we

excluded these cells from the ET experiments. Pig embryos

were derived in vivo or through parthenogenesis. A total of 167

embryo donors were used in this study, from which we collected

1,298 zygotes, 1,004 two-cell embryos and 91 morulae (Table

S5). Embryos were cultured in vitro until they reached the blasto-

cyst stage (Figures S4AA and S4B). Overall, 2,181 good quality

blastocysts with a well-defined ICM were selected for subse-

quent blastocyst injections, of which 1,052 were derived from zy-

gotes, 897 from two-cell embryos, 91 from morulae, and 141

from parthenogenetic activation (Table S5). We injected 3-10

hiPSCs into the blastocoel of each of these blastocysts (Figures

5A, S4A, and S4C; Table S6). After in vitro embryo culture, a total

of 2,075 embryos (1,466 for hiPSCs; Table S6; 477 for rodent

PSCs; Table S3) that retained good quality were transferred to

surrogate sows. A total of 41 surrogate sows received 30–50 em-

bryos each, resulting in 18 pregnancies (Table S6). Collection of

embryos between day 21-28 of development resulted in the har-

vesting of 186 embryos: 43 from 2iLD-hiPSCs, 64 from FAC-

hiPSCs, 39 from 4i-hiPSCs, and 40 from NHSM-hiPSCs (Figures

5B, S4A, S4D, and S4F). In addition, 17 control embryos were

collected from an artificially inseminated sow (Figure 5B).

Following evaluating the developmental status of the obtained

embryos, more than half showed retarded growth and were

smaller than control embryos (Figures 5B and S4B), as was

seen when pig blastocysts were injected with rodent PSCs (Fig-

ure 3B). Among different hiPSCs, embryos injected with FAC-

hiPSCs were more frequently found to be normal size (Figure 5C).

From the recovered embryos, and based on fluorescence imag-

ing (GFP for 2iLD-hiPSCs and FAC-hiPSCs; hKO for 4i-hiPSCs

and NHSM-hiPSCs), we observed positive fluorescence signal

(FO+) in 67 embryos among which 17 showed a normal size

and morphology, whereas the rest were morphologically under-

developed (Figures 5B). In contrast, among fluorescence nega-

tive embryos we found the majority (82/119) appeared normal

size (Figure 5E), suggesting contribution of hiPSCs might have

480 Cell 168, 473–486, January 26, 2017

interfered with normal pig development. Closer examination of

the underdeveloped embryos revealed that 50 out of 87 were

FO+ (Figures 5B). Among all the FO+ embryos the distribution

of normal size versus growth retarded embryos for each cell lines

was: 3:19 for 2iLD-hiPSCs, 7:14 for FAC-hiPSCs, 2:12 for 4i-

hiPSCs, and 5:5 for NHSM-hiPSCs (Figure 5D). Among normal

size embryos we found 3/13 from 2iLD-hiPSCs, 7/47 from

FAC-hiPSCs, 2/14 from 4i-hiPSCs, and 5/25 from NHSM-

hiPSCs that were FO+ (Figure 5B). All normal size FO+ embryos

derived from 2iLD-hiPSCs, 4i-hiPSCs, or NHSM-hiPSCs showed

a very limited fluorescence signal (Figure S5A). In contrast,

normal size FO+ FAC-hiPSC-derived embryos typically ex-

hibited a more robust fluorescence signal (Figures 6A and S5A).

Detecting fluorescence signal alone is insufficient to claim

chimeric contribution of donor hiPSCs to these embryos, as

auto-fluorescence from certain tissues and apoptotic cells can

yield false positives, especially when chimerism is low. We

thus sectioned all normal size embryos deemed positive based

on the presence of fluorescence signal and subjected them to

IHC analyses with antibodies detecting GFP or hKO. For 2iLD-

hiPSC-, 4i-hiPSC-, and NHSM-hiPSC-derived embryos, in

agreement with fluorescence signals observed in whole-embryo

analysis, we detected only a few hKO- or GFP-positive cells in

limited number of sections (Figure S5A). This precluded us

from conducting further IHC analysis using lineage markers.

For FAC-hiPSC-derived embryos, we confirmed via IHC analysis

(using an anti-GFP antibody) that they contained more human

cells (Figures 6A, S5A, and S5B). We then stained additional sec-

tions using antibodies against TUJ1, EPCAM, SMA, CK8, and

HNF3b (Figures 6B and S5C) and observed differentiation of

FAC-hiPSCs into different cell lineages. In addition, these cells

were found negative for OCT4, a pluripotency marker (data not

shown). Moreover, the presence of human cells was further veri-

fied with a human-specific HuNu antibody staining (Figure 6B)

and a sensitive genomic PCR assay using a human specific

Alu sequence primer (Figure 6C; Table S2). Together, these re-

sults indicate that naive hiPSCs injected into pig blastocysts inef-

ficiently contribute to chimera formation, and are only rarely

detected in post-implantation pig embryos. An intermediate

hPSC type (FAC-hiPSCs) showed better chimeric contribution

and differentiated to several cell types in post-implantation

human-pig chimeric embryos. It should be noted that the

levels of chimerism from all hiPSCs, including the FAC-hiPSCs,

in pig embryos were much lower when compare to rat-mouse

chimeras (Figures 1C, 1E, S1A, and 1B), which may reflect the

larger evolutionary distance between human-pig than between

rat-mouse.

DISCUSSION

Our study confirms that live rat-mouse chimeras with extensive

contribution from naive rat PSCs can be generated. This is in

contrast to earlier work in which rat ICMs were injected into

mouse blastocysts (Gardner and Johnson, 1973). One possible

explanation for this discrepancy is that cultured PSCs acquire

artificial features that make them more proliferative and/or better

able to survive than embryonic ICM cells, which in turn leads to

their more robust xeno-engraftment capability in a mouse host.

A

B C

D

E

Figure 5. Generation of Post-implantation Human-Pig Chimeric Embryos

(A) Schematic of the experimental procedures for the generation and analyses of post-implantation pig embryos derived from blastocyst injection of naive and

intermediate hiPSCs.

(B) Summary of the pig embryos recovered between day 21–28 of pregnancy.

(C) Bar graph showing proportions of normal size and growth retarded embryos, as well as the proportion of fluorescence-positive and -negative embryos,

generated from different types of hiPSCs.

(D) Bar graph showing the proportion of normal size and growth-retarded embryos (among those exhibiting a fluorescence signal) generated from different types

of hiPSCs.

(E) Bar graph showing the proportion of normal-sized and growth-retarded embryos (among those without exhibiting a fluorescence signal) generated from

different types of hiPSCs.

See also Figure S4 and Tables S5 and S6.

Rat-mouse chimeras generated by injecting donor rat PSCs into

a mouse host were mouse-sized and developed into adulthood

with apparently normal appearance and physiology. We further

show in this study that a rat-mouse chimera could live a full

mouse lifespan (about 2 years) and exhibit molecular signatures

characteristic of aged cells. This demonstrates that cells from

two different species, which diverged �18 million years ago, can live in a symbiotic environment and are able to support

normal organismal aging. The fact that rat PSCs were able to

contribute to the mouse gallbladder, an organ that is absent in

the rat, highlights the importance of embryonic niches in orches-

trating the specification, proliferation, and morphogenesis of

tissues and organs during organismal development and evolu-

tionary speciation (Izpisúa-Belmonte et al., 1992).

Previous interspecies blastocyst complementation experi-

ments generated host embryos by crossing heterozygous

mutant mouse strains, which were themselves generated

through targeted gene disruption in germline competent ESCs.

Cell 168, 473–486, January 26, 2017 481

(legend on next page)

482 Cell 168, 473–486, January 26, 2017

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-

bryos must 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.

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

(A) Representative bright field (left top) fluorescence (left bottom and middle) and i

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

(B) Representative immunofluorescence images showing chimeric contribution a

FAC-hiPSC derivatives are visualized by antibodies against GFP (top), TUJ1, SMA

magnification images of boxed regions. Scale bar, 100 mm.

(C) Representative gel images showing genomic PCR analyses of pig embryos d

and FAC-hiPSCs (surrogates #9159 and #18771) using a human specific Alu prim

with no genomic DNA loaded. pc, positive controls with human cells. Pig 1D, 1G

See also Figure S5 and Table S2.

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

bryos

mmunofluorescence (right) images of GFP-labeled FAC-hiPSCs derivatives in a

nd differentiation of FAC-hiPSCs in a normal size, day 28 pig embryo (FAC #1).

, CK8 and HuNu (middle). (Bottom) Merged images with DAPI. Insets are higher

erived from blastocyst injection of 2iLD-iPSCs (surrogates #8164 and #20749)

er. A pig specific primer Cyt b was used for loading control. nc, negative control

, and 1I, pig controls. ID, surrogate and pig embryos.

Cell 168, 473–486, January 26, 2017 483

optimized, may constitute a first step towards realizing the poten-

tial of interspecies blastocyst complementation with hPSCs. In

particular, they may provide a better understanding of human

embryogenesis, facilitate the development and implementation

of humanized animal drug test platforms, as well as offer new in-

sightsontheonsetandprogressionofhumandiseasesinaninvivo

setting. Ultimately, these observations also raise the possibility of

xeno-generating transplantable human tissues and organs to-

wards addressing the worldwide shortage of organ donors.

STAR+METHODS

Detailed methods are provided in the online version of this paper

and include the following:

d KEY RESOURCES TABLE

d CONTACT FOR REAGENT AND RESOURCE SHARING

d EXPERIMENTAL MODEL AND SUBJECT DETAILS

B Rodents

B Pigs

B Human iPSC Culture Media

B Culture and maintenance of rat ESCs/iPSCs and

mouse iPSCs

d METHOD DETAILS

B Chemicals Unless Otherwise Indicated, Chemicals

Were Obtained from Sigma-Aldrich

B Rat iPSC Generation

B Human iPSC Generation

B Generation of Fluorescently Labeled Rat PSCs and

hiPSCs

B Mouse Embryo Collection

B sgRNA Design and In Vitro Transcription

B Microinjection of Cas9 mRNA and sgRNAs to Mouse

Zygotes

B Microinjection of Rat PSCs to Mouse Blastocysts

B Mouse Embryo Transfer

B Genomic PCR

B Quantitative Genomic PCR

B Genotyping and DNA Sequencing

B Glucose Tolerance Test

B Cattle In Vitro Embryo Production

B Pig Parthenogenetic Embryo Production

B Microinjection of PSCs to Cattle and Pig Blastocysts

and Embryo Culture

B Pig and Cattle Blastocyst Immunostaining

B Pig In Vivo Embryo Recovery and Transfer

B Pig Parthenogenetic Embryo Transfer

B Immunocytochemistry

d QUANTIFICATION AND STATISTICAL ANALYSIS

SUPPLEMENTAL INFORMATION

Supplemental Information includes five figures and six tables and can be found

with this article online at http://dx.doi.org/10.1016/j.cell.2016.12.036.

AUTHOR CONTRIBUTIONS

J.W. and J.C.I.B. conceived the study. J.W. generated and characterized all

naive and intermediate hiPSC lines. K.S. generated and characterized primed

484 Cell 168, 473–486, January 26, 2017

hiPSCs. J.W. and T.H. generated rat iPSCs. J.W., A.P.-L., T.Y., M.M.V., D.O.,

A.O., P.R., C.R.E., J.W., and P.M.R. performed immunohistochemistry ana-

lyses of mouse and pig embryos. K.S., T.Y., E.S., A.P.-L., and M.M.V. per-

formed genotyping, genomic PCR, and genomic qPCR analyses. A.S., M.S.,

and J.P.L. performed mouse Cas9/sgRNA injection, blastocyst injection,

and embryo transfer. Y.S.B., M.S., and M.V. prepared hiPSCs, performed

morulae and blastocyst injections, and analyzed hiPSC contribution to cattle

and ppig ICMs. H.W. produced parthenogenetic pig embryos. D.A.S.,

Y.S.B., and M.V. produced cattle embryos. Work at UC Davis and University

of Murcia was performed under the supervision of P.J.R. and E.A.M., respec-

tively. E.A.M., M.A.G., C.C., I.P., C.A.M., S.S.B., A.N., and J.R. designed,

coordinated, performed, and analyzed data related to pig embryo collection,

embryo culture, blastocyst injection, embryo transfer, and embryo recover.

E.N.D., J.L., I.G., P.G., T.B., M.L.M.-M., and J.M.C. coordinated work

between Salk, and University of Murcia. J.W., P.J.R., and J.C.I.B. wrote the

manuscript.

ACKNOWLEDGMENTS

J.C.I.B. dedicates this paper to Dr. Rafael Matesanz, Director of the Spain’s

National Organ Transplant Organization. Rafael’s work has helped save thou-

sands of patients in need of an organ. He constitutes a relentless inspiration for

those of us trying to understand and alleviate human disease. The authors are

grateful to Xiomara Lucas, Maria Dolores Ortega, Moises Gonzalvez, Jose An-

tonio Godinez, and Jesus Gomis for their assistance throughout this work. We

thank the staff of the Agropor S.A. and Porcisan S.A. piggeries (Murcia, Spain)

for the help and excellent management of animals. We thank Joan Rowe, Bret

McNabb, Aaron Prinz, and Kent Parker and their crews for excellent assistance

with embryo transfers and pig care at UC Davis. We thank Mako Yamamoto for

help with mouse embryo dissection. We would like to thank Uri Manor of the

Salk Waitt Advanced Biophotonics Core for technical advice on imaging anal-

ysis. We would like to thank the Salk Stem Cell Core for providing cell culture

reagents. We would like to thank May Schwarz and Peter Schwarz for admin-

istrative help. We thank David O’Keefe for critical reading and editing of the

manuscript. This experimental study was supported by The Fundación Séneca

(GERM 19892/GERM/15), Murcia, Spain. The MINECO is acknowledged for

their grant-based support (BES-2013-064087 and BES-2013-064069) (to

C.A.M and A.N.). P.J.R was supported by a UC Davis Academic Senate

New Research grant. Work in the laboratory of J.C.I.B. was supported by

the UCAM, Fundacion Dr. Pedro Guillen, G. Harold and Leila Y. Mathers Char-

itable Foundation, and The Moxie Foundation.

Received: February 2, 2016

Revised: October 30, 2016

Accepted: December 22, 2016

Published: January 26, 2017

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