71 128
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 Vilarin˜ o,
3
Inmaculada Parrilla,
2
Delia Alba Soto,
3
Cristina A. Martinez,
2
Tomoaki Hishida,
1
Sonia Sa´ nchez-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 Nun˜ ez 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 Belmonte
1,8,
*
1
Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA
2
Department of Animal Medicine and Surgery, University of Murcia Campus de Espinardo, 30100 Murcia, Spain
3
Department of Animal Science, University of California Davis, One Shields Avenue, Davis, CA 95616, USA
4
Universidad Cato´ lica San Antonio de Murcia (UCAM) Campus de los Jero´ nimos, N 135 Guadalupe 30107 Murcia, Spain
5
Clinica Centro Fundacio´ n Pedro Guille´ n, Clı´nica CEMTRO, Avenida Ventisquero de la Condesa 42, 28035 Madrid, Spain
6
Hospital Clı´nico de Barcelona-IDIBAPS, Universitat de Barcelona, 08007 Barcelona, Spain
7
Present address: Graduate School of Agriculture, Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi,
Nara 631-8505, Japan
8
Lead Contact
*Correspondence:
belmonte@salk.edu http://dx.doi.org/10.1016/j.cell.2016.12.036SUMMARY
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 naı¨ve 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
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2017 Elsevier Inc.
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