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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
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