Viable Rat-Mouse Chimeras Where Do We Go from Here

and supported by,the phenotypic PCP features of Frizzled/PCP core group genes on one side and that of the Fat/ Dachsous system on the other.Flies carrying mutations in Frizzled/PCP core proteins exhibit defects in PCP throughout the wing.In contrast,the Fat/Dachsous system mainly affects polarity in the proximal half of the wing, as this area strongly depends on cellular realignment and rotation during the switch to the proximal-distal PCP axis. Together,these observations provide an exciting new framework for under-standing the generation of PCP and its relation to new mechanisms that sculptthe shape of organs in general.REFERENCESAigouy,B.,Farhadifar,R.,Staple,D.B.,Sagner,A.,Ro¨per,J.,Ju¨licher,F.,and Eaton,S.(2010).Cell142,this issue,773–786.Baena-Lo´pez,L.A.,Baonza,A.,and Garcı´a-Bel-lido,A.(2005).Curr.Biol.15,1640–1644.Classen,A.K.,Anderson,K.I.,Marois,E.,and Ea-ton,S.(2005).Dev.Cell9,805–817.Harumoto,T.,Ito,M.,Shimada,Y.,Kobayashi,T.J.,Ueda,H.R.,Lu,B.,and Uemura,T.(2010).Dev.Cell19.Published online September2,2010.10.1016/j.devcel.2010.08.004.Lawrence,P.A.,Struhl,G.,and Casal,J.(2007).Nat.Rev.Genet.8,555–563.Seifert,J.R.,and Mlodzik,M.(2007).Nat.Rev.Genet.8,126–138.Shimada,Y.,Yonemura,S.,Ohkura,H.,Strutt,D.,and Uemura,T.(2006).Dev.Cell10,209–222.Strutt,D.(2003).Development130,4501–4513.Vinson,C.R.,and Adler,P.N.(1987).Nature329,549–551.Wu,J.,and Mlodzik,M.(2009).Trends Cell Biol.19,295–305.Viable Rat-Mouse Chimeras:Where Do We Go from Here?Davor Solter1,*1Institute of Medical Biology,A*STAR,138648Singapore,Republic of Singapore*Correspondence:davor.solter@.sgDOI10.1016/j.cell.2010.08.021In a tour-de-force study,Kobayashi et al.(2010)describe thefirst viable rat-mouse chimeras and demonstrate that rat induced pluripotent stem(iPS)cells can rescue organ deficiency in mice. Rat iPS cells formed a fully functional pancreas when injected into mouse blastocysts lacking the Pdx1gene required for pancreas formation.Experimentally produced chimeras between different mouse strains(Tarkow-ski,1961)have been an exceedingly useful tool for developmental biologists, contributing to our understanding of the establishment of cell lineages,cell deter-mination,and the development of the immune system and other organs.In this issue of Cell,Kobayashi et al.(2010) dramatically extend the potential of mam-malian chimeras with their report of viable rat-mouse chimeras that can develop to term and become fully functional adults. In their study,Kobayashi and col-leagues relied on previous knowledge but also added a few new wrinkles.They first derived mouse and rat embryonic stem(ES)cells and induced pluripotent stem(iPS)cells using standard methods for the mouse and capitalizing on therecent isolation of rat ES and iPS cells(Buehr et al.,2008;Li et al.,2008).Bothmouse and rat cells were tagged withdifferentfluorescent dyes,allowing theauthors to follow their distribution in thedeveloping chimeras.The authors wantedto prove,at least in principle,that xenoge-neic organ complementation could beachieved,that is,that donor cells of onespecies could rescue a defect in organdevelopment in a recipient of a differentspecies.So,as afirst step,they set outto produce viable chimeras between ratsand mice,even though many previousefforts to make such chimeras had failed.The only viable intergeneric chimera—that is,a hybrid between animals fromdifferent genera—reported so far isthe geep between a sheep(Ovis aries)and a goat(Capra hircus)(Fehilly et al.,1984).To test the possibility that viable rat-mouse chimeras could be formed,Kobayashi et al.(2010)injectedfluores-cently labeled mouse or rat iPS cells intorat or mouse blastocysts,respectively,and returned them to blastocyst-compat-ible pseudopregnant females(that is,foster mothers of the same species asthe blastocysts).The authors then exam-ined the resulting fetuses,newborns,andadults and found evidence of a substantialcontribution of donor stem cells to tis-sues and organs of the host(Figure1A).Despite a big contribution of donor cells,the size of newborn and adult chimeras(with one exception)was determined by676Cell142,September3,2010ª2010Elsevier Inc.the species of the host blastocyst.It is not clear whether it is the embryo itself or the uterine environment that determines the extent of chimera growth.To distinguish between these possibilities,one would have to transfer chimeric embryos into the uterus of pseudopregnant females of the same species as the donor stem cells (not the blastocysts).Previous studies suggest that such experiments would fail because of the need for compatibility between the fetal part of the placenta and the uterus (Rossant et al.,1982).Besides controlling the size and growth of the chimera,the host blastocyst seems to impose additional morphogenetic reg-ulation.The postimplantation develop-ment of normal rat and mouse embryos is very similar,but there are differences in organ morphogenesis.One of the most noticeable differences is the pres-ence of a gall bladder in mice and its absence in rats.In all adult chimeras produced by combining rat stem cells and mouse blastocysts,the resulting ‘‘mouse-like’’chimeras had a gall bladder despite the significant contribution of rat cells to abdominal organs.Reciprocal chimeras were ‘‘rat-like’’and,again,despite a significant contribution from mouse cells to abdominal organs,the gall bladder was absent.These results suggest that cells of the blastocyst inner cell mass possess a ‘‘morphogenetic’’capacity that controls the behavior of injected stem cells at all developmental stages.This may explain why Kobayashi et al.were able to successfully inject rat stem cells into mouse blastocysts,whereas insertion of the rat inner cell mass into the mouse blastocyst cavity did not result in viable rat-mouse chimeras (Gardner and Johnson,1973).This notion could be tested further using tetraploid comple-mentation (that is,donor ES or iPS cells are injected into tetraploid blastocysts)to produce rat-mouse chimeras (Nagy et al.,1993).Tetraploid blastocyst cells cannot participate in formation of the embryo proper;thus,the resulting fetus (and adult)is derived entirely from the injected cells,whereas the placenta and extraembryonic membranes are derived from the tetraploid blastocyst.It remains to be seen whether this approach could produce a fetus derived entirely from mouse ES cells after their injection into a rat tetraploid blastocyst that then develops in the uterus of a pseudopreg-nant rat female.A major goal of the Kobayashi et al.study was to determine whether stem cells from a xenogeneic donor mammal could correct a genetic defect in a recipient mammal of a different species.So,in their next set of experiments,the authors in-jected rat iPS cells into recipient mouse blastocysts that lacked the Pdx1gene,Figure 1.Generating Rat-Mouse Chimeras(A)Induced pluripotent stem (iPS)cells were derived from adult mouse and rat cells and were labeled with different fluorescent proteins.Rat (blue)and mouse (red)iPS cells were injected into reciprocal blastocysts (mouse into rat and vice versa)to produce intergeneric chimeras.From these blastocysts,several chimeras were born and some survived to adulthood.The contribution of injected donor stem cells was observed throughout the body of the host.The size and morphology of the newborn and adult chimeras was determined by the host blastocyst.(B)Fluorescently labeled rat iPS cells (blue)were injected into normal mouse blastocysts (left)or blastocysts lacking the Pdx1gene (right),which encodes the transcription factor pancreatic and duodenal homeobox 1that is required for pancreas development.Chimeras derived from normal or Pdx1-deficient mouse blastocysts showed an extensive contribution of rat cells to all tissues.However,in the Pdx1-deficient chimeras,the entire pancreas was derived from donor rat cells (inset,blue)and was fully functional,including production of insulin by b islet cells.Cell 142,September 3,2010ª2010Elsevier Inc.677which encodes a transcription factor (pancreatic and duodenal homeobox1) that is essential for development of the pancreas and formation of insulin-producing b islet cells.Although they observed a substantial contribution of rat cells to different organs and tissues,most importantly,the pancreas of the rat-mouse chimeras was composed exclusively of rat cells(Figure1B).Thus,cells derived from rat iPS cells were able to completely rescue the genetic deficiency of the host mouse blastocyst.These rat-mouse chimeras developed into adult animals with a normal functional pancreas,demon-strating that xenogeneic organ comple-mentation is achievable.This is a remark-able accomplishment.So where do we go from here?Although human ES and iPS cells offer hope for tissue and cell replacement therapies in the not too distant future,the replacement of complex organs—lung,kidney,liver, gut,and,of course,pancreas—is likely to be much more difficult.Several strategies for organ replacement are being tested. These include the growth of organs in vitro with mixtures of different stem cells and biocompatible scaffolds or the gener-ation of‘‘humanized’’pigs engineered to lack certain antigens so that their organs can be used for transplantation in human patients with a reduced chance of immune rejection.Could production of human organs in,for example,human-pig chimeras be an alternative approach? Although production of viable rat-mouse chimeras could be viewed as afirst step in this direction,as Kobayashi et al. propose,there are huge biological and technical challenges.For example,themouse and rat are developmentally verysimilar(apart from size),but it is not clearthat chimeras between animals belongingto different phylogenetic families or orderswould be viable.Indeed,the only attemptsto make such chimeras(between a mouseand a bank vole)have failed(Mystkowska,1975).In this experiment,the mouse-bankvole chimeras were made by aggregationof embryos;it is possible that injection ofbank vole stem cells into mouse blasto-cysts,followed by their developmentin the uterus of mouse foster mothersmight yield positive results.Successfulchimerism between members of differentorders(the pig and human,for example)seems very unlikely,and attempts toproduce early postimplantation human-mouse chimeras have not been encour-aging(James et al.,2006).Even if wesucceed in developing organ-deficientpigs by genetic manipulation and pro-ducing pig-human chimeras in which theparenchymal cells of the specific organare entirely derived from human cells,immune rejection will still be a problembecause the human organ carried by thepig will contain pig-derived stromal cellsand blood vessels.Finally,there are huge legal and ethi-cal barriers to creating human-animalchimeras and,indeed,their production isforbidden in most countries.However,itis possible that injecting human ES oriPS cells into a mouse blastocyst and al-lowing limited(early postimplantation)development of human-mouse chimeraswould be approved for the specificpurpose of testing the differentiationpotential of human stem cells.Yet suchexperiments will be complicated,timeconsuming,difficult to interpret,and,Isuspect,will never become part of thestandard protocols regulating the medicaluse of human stem cells.Although xeno-geneic organ complementation is unlikelyto be a viable strategy for regenerativemedicine,the elegant work of Kobayashiet al.is a boon for researchers seekingto better understand the biology of stemcells and mammalian development.REFERENCESBuehr,M.,Meek,S.,Blair,K.,Yang,J.,Ure,J.,Silva,J.,McLay,R.,Hall,J.,Ying,Q.-L.,and Smith,A.(2008).Cell135,1287–1298.Fehilly,C.B.,Willadsen,S.M.,and Tucker,E.M.(1984).Nature307,634–636.Gardner,R.L.,and Johnson,M.H.(1973).Nat.NewBiol.246,86–89.James,D.,Noggle,S.A.,Swigut,T.,and Brivanlou,A.H.(2006).Dev.Biol.295,90–102.Kobayashi,T.,Yamaguchi,T.,Hamanaka,S.,Kato-Itoh,M.,Yamazaki,Y.,Ibata,M.,Sato,H.,Lee,Y.-S.,,Usui,J.-i.,Knisely,A.S.,et al.(2010).Cell142,this issue,787–799.Li,P.,Tong,C.,Mehrian-Shai,R.,Jia,L.,Wu,N.,Yan,Y.,Maxson,R.E.,Schulze,E.N.,Song,H.,Hsieh,C.-L.,et al.(2008).Cell135,1299–1310.Mystkowska,E.T.(1975).J.Embryol.Exp.Mor-phol.33,731–744.Nagy, A.,Rossant,J.,Nagy,R.,Abramow-Newerly,W.,and Roder,J.C.(1993).Proc.Natl.A90,8424–8428.Rossant,J.,Mauro,V.M.,and Croy,B.A.(1982).J.Embryol.Exp.Morphol.69,141–149.Tarkowski,A.K.(1961).Nature190,857–860.678Cell142,September3,2010ª2010Elsevier Inc.。