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A
Day 5
Day 8
Day 12
Day 12
Beating
activity
Beating
activity
Day 0
500 µm
15 µm
α-actinin
Fluo-4
B
Day 0
4 weeks
4 weeks
4 weeks
C
0 h
24 h
Integrated
iPS
iPS
head
tail
heart
morulae
Day 0
blastocyst
blastocyst
LacZ
9.5 dpc
LacZ
FIGURE 6.3
Imaging iPS cell differentiation from in vitro analysis to chimeric offspring. (A) Differentiating
iPS cells in vitro produces day 5 embryoid bodies capable of multilineage differentiation, with
bona fide cardiogenesis demonstrated by beating activity at day 8, expression of cardiac sarco-
meric proteins (middle panels), and functional excitation-contraction coupling demonstrated
by calcium transients (far right panel). (B) Injection of iPS cells into the subcutaneous tissue
of immunodeficient host produces engraftment of labeled progeny detected by biolumines-
cence 4 weeks after transplantation. The stable engraftment enables teratoma formation to
depict trilineage differentiation in vivo and to validate pluripotent differentiation capacity
of iPS cells. (C) Transplantation of labeled iPS cells into host morula demonstrates the func-
tional equivalency of iPS cells compared to native blastomeres as the chimeric tissue develops
through gastrulation and gives rise to early-stage embryos. The growth of iPS cell-derived
chimeric embryos can be monitored in real time with in vivo imaging according to luciferase
expression within pregnant mothers throughout development and confirmed with lacZ stain-
ing (far right panels).
and endoderm lineages, as well as the persistence of poorly differentiated
cytotypes. Together, these experimental systems documented the multiple
tissues derived from in vivo differentiation and spontaneous formation of
complex cytoarchitecture derived from iPS cell clones.
Ultimately, normal differentiation of stem cells is validated in a devel-
opmental model system that tests the ability of progenitor cells to function
equivalently to native embryonic stem cells. This can be done through either
blastocyst injection or aggregation techniques. The chimeric offspring can
be directly analyzed for proper tissue-specific differentiation or used to
determine germline transmission. The highest stringency for pluripotency
from iPS cells has been documented by tetraploid aggregation in which the
entire embryo proper is derived from transplanted stem cells within a host
embryonic environment that has been compromised by electrofusion at the
two-cell stage to prevent native blastomeres from developing into embryonic
tissue [42,43]. Thus, the resulting embryo that develops and gives rise to adult
 
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