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Cell Stem Cell
Brief Report
iPS Cells Can Support Full-Term Developmentof Tetraploid Blastocyst-Complemented Embryos
Lan Kang,
1,2
Jianle Wang,
2
 Yu Zhang,
2
Zhaohui Kou,
2
and Shaorong Gao
2,
*
1
Graduate Program, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China
2
National Institute of Biological Sciences, Beijing 102206, China*Correspondence:gaoshaorong@nibs.ac.cnDOI 10.1016/j.stem.2009.07.001
To our knowledge, for the first time, wedemonstrate that induced pluripotentstem cells (iPSCs) can autonomouslygeneratefull-termmiceviatetraploidblas-tocysts complementation. Differentiatedsomatic cells can be reprogrammed intoiPSCs by forced expression of four tran-scription factors—Oct4, Sox2, Klf4, andc-Myc. However, it has been unclearwhether reprogrammed iPSCs are fullypluripotent, resembling normal embryonicstem cells (ESCs), as no iPSC lines haveshown the ability to autonomouslygenerate full-term mice after injection intotetraploid blastocysts. Here we provideevidence demonstrating that an iPSC lineinduced by the four transcription factorscan be used to generate full-term micefromcomplementedtetraploidblastocystsand thus appears to be fully pluripotent.This work serves as a proof of principlethat iPSCs can in fact generate full-termembryos by tetraploid complementation.Reprogramming of differentiated so-matic cells into iPSCs via expression of four defined transcription factors has at-tracted great scientific and public atten-tion, and the generation of iPSCs fromindividual patients has raised hopes fortreating many degenerative and geneticdiseases ( Dimos et al., 2008; Ebert et al.,2009;Hannaetal.,2007;Parketal.,2008;Soldneretal.,2009;Takahashietal.,2007;Takahashi and Yamanaka, 2006; Werniget al., 2008; Yu et al., 2007 ). iPSCs canfunctionally differentiate into all cell line-ages within chimeric mice, includinggerm cells that can ultimately give rise tooffspring ( Maherali et al., 2007; Okita et al.,2007; Takahashi and Yamanaka, 2006;Wernig et al., 2007 ). However, unlike ESCsor even somatic cell nuclear transfer(SCNT)-produced NT-ESCs, no iPSC lineshave so far fulfilled the most stringentpluripotency criterion of being able togenerate full-term mice via tetraploidblastocystcomplementation( HayashiandSurani, 2009; Jaenisch, 2008; Maheraliand Hochedlinger, 2008; Wernig et al.,2007 ). To understand the reprogramm-ing mechanisms that occur during iPSCgeneration, it is important to addresswhether iPSCs are fully pluripotent. As in previous reports ( Brambrink et al.,2008; Stadtfeld et al., 2008 ), doxycycline(Dox)-controlled Tet-on-inducible lentivi-ruses carrying cDNAs of the transcriptionfactors Oct4, Sox2, Klf4, and c-Myc weretransduced into mouse embryonic fibro-blast (MEF) cells from ROSA26-M2rtTA transgenic mice ( Figure 1 A). After viraltransduction, the MEFs were cultured inES culture medium containing Dox for 12days until ES-like colonies appeared,and then the Dox was removed from theculture medium ( Figure 1B). Four dayslater, ES-like colonies were individuallydigested and replated in 24-well plates. After propagation, we selected fiveiPSC lines that exhibited typical ESCmorphology ( Figure 1C) for long-termculture and characterization. The ES-likecolonies generated were positive for APactivity (seeFigure S1available online).RT-PCR and qPCR analysis demon-stratedthattheexpressionofpluripotencymarker genes, including Oct4, Sox2, andNanog, was comparable across all fiveiPSC lines and normal ESC lines butshowed dramatic differences relative toMEFs( Figures1Dand1E).Silencingoftheexogenous lentiviral vectors was alsoobserved ( Figure S2 ). Immunofluorescentstaining further confirmed the proteinexpression of three transcription factorsand the surface marker SSEA-1 in theiPSCs ( Figure 1F). In addition, other pluri-potency-relatedgeneswereallexpressedat similar levels in the iPSCs and normalESCs ( Figure S3 ). The karyotypes of allfive iPSC lines were normal, with 40 chro-mosomes ( Figure S4 ). Southern blot anal-ysis was used to examine viral integrationinto the genome of iPSCs and indicatedthat there were one or two integrationsfor each of the four lentiviral constructsinto the iPS genome ( Figure 1G).To investigate the pluripotency of thegenerated iPSCs, we performed in vitrodifferentiation and teratoma and chimeraexperiments. The iPSCs formed EBs effi-cientlyinvitro,andupregulationofmarkergenes for all three germ layers was de-tected in the plated EBs ( Figure S5 ). Wethen transplanted the iPSCs subcutane-ously into immune-deficient SCID miceand found that all the iPSC lines testedgenerated teratomas 3–4 weeks aftertransplantation. Hematoxylin and eosin(H&E) staining indicated that the iPSCscould differentiate into a variety of celltypes from all three germ layers ( Fig-ure 1H). The LiPS-6 line generated post-natal chimeras with normal appearancethat gave rise to germline offspring aftermating with ICR female mice ( Figure 1I).However, we could not produce full-termmice from this line via tetraploid comple-mentation. Meanwhile, we noticed a veryhigh degree of chimerism in the chimerasgenerated from one iPSC line, LiPS-8, asindicated by its chimera coat color, whichwas almost completely contributed byiPSC ( Figure 1J). We therefore decided totest whether these iPSCs were capableofgeneratingfull-termmicethroughtetra-ploid complementation.We first generated tetraploid embryosby fusing late two-cell-stage embryos toone-cell-stage embryos using an ElectroCellManipulator(BTX2001).Theembryoswere collected from B6D2F1 female micemated with male mice of the same strain.Ten to fifteen iPSCs were microinjectedinto the cavity of tetraploid blastocysts,and the complemented blastocysts (n =200) were transferred into seven pseudo-pregnant ICR female mice after 2–3 hr of recovery. On the due date, a C-sectionwas performed to ascertain whether anyof the tetraploid-complemented embryos
Cell Stem Cell
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, August 7, 2009
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2009 Elsevier Inc.
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Please cite this article in press as: Kang et al., iPS Cells Can Support Full-Term Development of Tetraploid Blastocyst-Complemented Embryos, CellStem Cell (2009), doi:10.1016/j.stem.2009.07.001
 
haddevelopedintoliveanimals.AsshowninFigure 2 A, we produced two full-termmice from the iPSC-complemented tetra-ploid blastocysts. One pup ( Figure 2 Ab)was overweight (2.72 g) and died within1 hr following the C-section. The otherpup ( Figure 2 Aa) was of normal weight(1.36 g) and survived for 3 days but diedbecause the surrogate mother refused tofeed it. To test whether the fully iPSC-derived mouse can survive to adulthood,we repeated this experiment with tetra-ploid blastocysts from ICR mice. Aftertransplantation of 187 tetraploid-comple-mented blastocysts into the oviducts of sevenpseudopregnantmice,wesuccess-fully produced two more full-term pupsfrom these embryos ( Figure 2 A), and onepup survived into adulthood and appearsto be healthy ( Figure 2B). The efficiencyof generating full-term mice from iPSC-complemented ICR tetraploid embryoswas similar to the B6D2F2 backgroundembryos(1.07%versus1.00%,Figure2F).We then performed SSLP and rtTA anal-ysisoftheorganscollectedfromthethreedead pups and the tail tip of the livemouse. The results clearly indicated thatall the pups were derived from the iPSCs(mostly C57BL/6 background) and notfrom the tetraploid embryos (B6D2F2 orICR background) ( Figures 2C–2E). More-over, we successfully cultured skin fibro-blast cells from the pups that died shortlyafter the C-section. After adding Dox to
Figure 1. Generation ofInducible iPSCsandCharacterization
(A) Morphology of MEF cells collected fromROSA26-M2rtTA transgenic mice.(B) Colonies formed after infection and Doxinduction.(C) Morphology of iPSCs after propagation.(D) RT-PCR revealed that inducible iPSCsexpressed ESC marker genes including
OCT4
,
SOX2
, and
NANOG
.(E) qPCR revealedthat the expression level of ESCmarkergenesiniPSCswassimilartothatofnormalESCs.(F) Immunofluorescent staining results demon-strated that the iPSCs were positive for OCT4,SOX2, NANOG, and SSEA-1. Scale bars, 20
m
m.(G) Southern blot analysis showed that there wereone or two integrations for each virus into the iPSgenome.(H) Teratomas formed 4 weeks after injection of iPSCs into SCID mice. Tissues of the three germlayers were detected by H&E staining.(I) The chimeric mouse showed germline transmis-sion capability.(J) High chimerism (>90%) was found in thechimeric mice produced from a particular iPSCline (LiPS-8).
Cell Stem Cell
Brief Report
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Cell Stem Cell
5
, August 7, 2009
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2009 Elsevier Inc.
Please cite this article in press as: Kang et al., iPS Cells Can Support Full-Term Development of Tetraploid Blastocyst-Complemented Embryos, CellStem Cell (2009), doi:10.1016/j.stem.2009.07.001
 
the medium, many ES-like colonies ap-peared ( Figure S6 ), and the efficiencywas substantially higher than for the firstround of iPS induction. As the inducibleiPSC line LiPS-8 is, to our knowledge,the first iPSC line that has been usedsuccessfully to generate mice by tetra-ploid complementation, we investigatedthe gene expression profile of this partic-ular cell line as compared with the normalESClines,R1,andtheparentalMEFcells.The data clearly showed that the geneexpression profile of this specific iPS lineis very similar to the normal ESCs ( Fig-ure 2G). Meanwhile, bisulfite genomicDNA sequencing analysis of the Oct4promoter showed that it is highly unme-thylated in the inducible iPSC line LiPS-8,whereas it is highly methylated in theparental MEF cells ( Figure S7 ).In conclusion, we have successfullyproduced full-term mice from iPSC-complemented tetraploid blastocysts. Although these findings are an importantproof of principle, it would be prematureto make claims based on them aboutwhether iPSCs in general are functionallyequivalent to normal ESCs, as we havehere only identified one iPSC line thatpossesses this capacity, and the processis also very inefficient. It is possible thatthis line has specific characteristics thatallow tetraploid-complementation activitybut are not shared by other iPSC lines. Itwill be interesting to evaluate whetheriPSCs generated using fewer factors ordifferent approaches are capable of pro-ducing tetraploid-complemented mice( Kim et al., 2008, 2009; Nakagawa et al.,2008 ) to assist with advancing overallunderstanding of the molecular mecha-nisms involved in iPSC generation. More-over, partial reprogramming could poten-tially provide an explanation for theinefficient differentiation of iPSCs intocertain cell lineages, and obtaining morefully reprogrammed iPSCs in humansmight facilitate directed differentiation of iPSCs into ideal cell types.
 ACCESSION NUMBERS
Microarray data can be assessed at Gene Expres-sion Omnibus ( http://www.ncbi.nlm.nih.gov/geo/  )under accession number GSE17004.
SUPPLEMENTAL DATA 
Supplemental Data include Supplemental Experi-mental Procedures, Supplemental References,
Figure 2. Generation of iPS-Tetraploid Mice
(A) Four pups were produced autonomously from iPSCs complemented with tetraploid blastocystscollected from either B6D2F1 mice or ICR mice. One pup from iPS-B6D2F1 tetraploid complementationdied 3 days after C-section because the surrogate mother refused feeding (Aa), one overweight pup fromiPS-B6D2F1 tetraploid complementation died (Ab), one pup from iPS-ICR tetraploid complementationdied shortly after C-section (Ac), and one pup from iPS-ICR tetraploid complementation survived (Ad).(B) The pup (Ad) generated from iPS-ICR tetraploid complementation grows into an adult.(C) SSLP analysis of organs of three dead tetraploid-complemented pups (Aa–Ac). D6Mit15 andD11Mit236 were used.(D) Genomic PCR analyzed the transgene, rtTA, in the organs of three dead tetraploid-complementedpups.(E) Genomic PCR and SSLP were performed to analyze the live adult mouse derived from iPS-tetraploidcomplementation. Transgene rtTA and D11Mit236 were analyzed, respectively.(F) Efficiency of generation of iPSC-tetraploid-complemented pups. The efficiency is lower than theefficiency of using normal B6D2F1 ESCs ( Table S1 ).(G) Scatter plots comparing global gene expression patterns between iPSCs and ESCs (left), betweeniPSCs and MEFs (middle), and between R1 and MEFs (right), as determined by DNA microarray. ‘‘R’’ indi-cates Pearson correlation coefficient, and the lines indicate 2-fold changes in gene expression levelsbetween the samples.
Cell Stem Cell
5
, August 7, 2009
ª
2009 Elsevier Inc.
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Cell Stem Cell
Brief Report
Please cite this article in press as: Kang et al., iPS Cells Can Support Full-Term Development of Tetraploid Blastocyst-Complemented Embryos, CellStem Cell (2009), doi:10.1016/j.stem.2009.07.001
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