Generation of Rat Pancreas in Mouse
by Interspecific Blastocyst Injection
of Pluripotent Stem Cells
Kobayashi et al. 2010 Cell, Vol. 142, 787-799
TD Décembre 2010
1-Generation of Donor Mouse iPSC-Derived Pancreas
in Pdx1-Deficient Neonates (Figure 1)
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Pdx1-/- blastocyst complementation
Blastocysts obtained by an intercross of heterozygote Pdx1-LacZ knock-in mice
(Pdx1+/-)
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with GT3.2 iPSCs (miPSCs) generated from tail tip fibroblasts (TTFs) of an adult
eGFP transgenic C57BL6J mouse using 3 factors (Oct3/4, Sox2, Klf4)
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with G4.2 mESCs
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Neonates highly chimeric
Pancreas present in all neonates regardless of host genotype (including Pdx1-/-)
Both miPSCs and mESCs supplied all pancreatic cell lineages (exocrine and
endocrine tissue)
2-miPSCs Rescued Pdx1-/- Mice (adult) by Blastocyst
Complementation (Figure 2)
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Question? Can mPSCs rescue Pdx1-/- lethality via blastocyst complementation?
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Pdx1-/- chimeric mice survive to adulthood
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Pdx1-/- chimeric mice can even serve as founders, transmitting their genotype to the
next generation
Mating Pdx1-/- founder male mice with Pdx1+/- female mice increased to 50% the
proportion of Pdx1-/- pup
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In both Pdx1-/- and Pdx1+/- mice injected with eGFP-miPSCs, miPSC-derived cells
contributed to all tissues of the body
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Glucose tolerance tests
3-Transplantation of miPSC-Derived Islets Corrected
Hyperglycemia in Diabetic Mice
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Blastocyst complementation permits both:
– Pancreas generation
– Diabetic therapy using donor iPSC-derived syngenic islets
4-Generation of Interspecific Chimeras between Mouse
and Rat (Figure 4A)
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Goal: to generate interspecific chimeras between
mouse and rats
1) Generation of mouse iPSCs (eGFP+)
2) Generation o rat iPSC (eGFP+) and rat ESCs (3i
medium)
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Because post-implantation is severely impaired
after intra-uterine transfer of xenogenic blastocysts,
injected r-blastocysts or m-blastocysts were
transfered into the uteri of pseudopregnant rats or
mice respectively.
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eGFP expressing cells are found in the body of
each injected conceptus but never found in the
placenta.
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Successfull generation of interspecific chimeras
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Quantification of mouse or rat iPSCs to chimera
tissues (varyinf from organ-to-organ and from
individual-to-individual
Both embryonic development (mouse E13.5 and rat E15.5) and
degree of chimerism are lower in interspecific chimeras than
in intraspecific chimeras
5-Interspecific Chimeras Were Live-born; Some Grew
into Adulthood
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Analysis at neonatal and adult stages
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Chimerism can be judged by coat color because miPSCs (C57BL/6, black coat) were
injected in rat blastocyst (Wistar, white coat) or riPSCs (Wistar) were injected in
mouse blastocysts (BDF11 x C57BL/6, black coat)
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20% full term development rate of interspecific chimeras either with miPSCs in rat
blastocysts or riPSC in mouse blastocysts
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50% full term development rate of intraspecific chimeras 50%
8 weeks of age chimeras
6-Determination of Body Size in Interspecific Chimeras
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Question: What determines the size of interspecific chimeras?
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Adult rats = 10X bigger than adult mice
Newborn rats = 3X bigger than newborn mice
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Because mouse and rat gestation times are of similar length (19 and 21 days)
organogenesis resquires more cell proliferation and differentiation during rat
development than during mouse development.
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In most chimeras, the sizeof the newborns seemed to conform with that in the
species from which the blastocyst was derived (when the degree of chimerism is low)
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Correlation between body weight and contribution of donor iPSC derived cells as
estimated from hair color and peripheral blood cells
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The origin of placenta and uterus may also have a key role in size determination.
Chimerism 5.35%
15.6%
17.8%
55.5%
7-Distribution of Donor iPSC-Derived Cells
in the Xenogenic Environment
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Goal: to determine the distribution of mouse or rat-iPSC-derived eGFP positive cells
in neonatal interspecific chimeras
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Rats do not have gallbladders whereas mice do.
Rat interspecific chimeras do not have gallbladders but mouse interspecific chimeras
have
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miPSC and riPSC contribute to the germ line in intraspecific chimeras but do not in
interspecific chimeras.
8-Generation of Rat Pancreas in Mouse via Interspecific
Blastocyst Complementation
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Goal: to generate a xenogeic rat pancreas in Pdx1-/- mice by interspecific blastocyst
complementation
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Selection of a very efficient rat iPSC line (riPSC#3) for embryonic developmental rate
and degree of chimerism after injection in mouse embryos.
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139 injected Pdx1-/- blastocysts / 34 mice born analyzed at neonatal and adult
stages.
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The pancreas of Pdx1-/- interspecific mouse chimeras is entirely derived from riPSCs
cells (confirmed by FACS analysis, genotyping and immunohistochemistry)
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In adult mice, intact pancreas (functional test OK)
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Generation of a xenogenic iPSC-derived organ is possible via interspecific blastocyst
complementation