Livestock Induced Pluripotent Stem Cells

Y Lu1,2, JL Mumaw1, FD West1 and SL Stice1

1Department of Animal and Dairy Science, Regenerative Bioscience Center, University of Georgia, Athens, GA, USA; 2State Key Laboratory forConservation and Utilization of Subtropical Agro-Bioresources, Animal Reproduction Institute, Guangxi University, Nanning, Guangxi, China

Contents

Chimeric animals generated from livestock-induced pluripo-tent stem cells (iPSCs) have opened the door of opportunity togenetically manipulate species for the production of biomed-ical models, improving traits of agricultural importance andpotentially providing a system to test novel iPSC therapies.The potential of pluripotent stem cells in livestock has longbeen recognized, with many attempts being chronicled toisolate, culture and characterize pluripotent cells from embryos.However, in most cases, livestock stem cells derived fromembryonic sources have failed to reach a pluripotent statemarked by the inability to form chimeric animals. The in-depthunderstanding of core pluripotency factors and the realizationof how these factors can be harnessed to reprogram adult cellsinto an induced pluripotent state has changed the paradigm oflivestock stem cells. In this review, we will examine theadvancements in iPSC technology in mammalian and avianlivestock species.

Introduction

The discovery of mouse embryonic stem cells (mESCs)in combination with new mammalian gene-alteringtechnologies (Slightom et al. 1980; Folger et al. 1982)led to the first gene knockout mice (Koller et al. 1989;Thompson et al. 1989; Zijlstra et al. 1989). Theseadvances in biomedical research allowed for the forma-tion of mice which modelled complex diseases generat-ing groundbreaking discoveries in the role of thegenetics behind these diseases. Genetically engineeredmice have come to be indispensible models for findingnovel therapeutics and drug treatments, but they areoften unsuitable to accurately model human diseases.

The isolation and culture of pluripotent stem cells,cells capable of forming any cell type of the body, fromembryos remained an elusive feat until 1981 when twogroups successfully developed a co-culture system thatwas capable of maintaining mESCs in a pluripotentstate with feeder cells secreting necessary factors tomaintain pluripotency (Evans and Kaufman 1981;Martin 1981). These first mESC lines demonstratedcharacteristics that have come to define ESCs in allspecies. ESCs can be infinitely expanded, differentiatedinto all three germ layers (endoderm, ectoderm andmesoderm) in vitro as embryoid bodies (EBs) and in vivoin the form of teratomas (Evans and Kaufman 1981;Martin 1981), contributed to all tissue types includingthe germline in chimeric animals (Bradley et al. 1984)and acted as the inner cell mass (ICM) in tetraploidcomplementation assays (Eggan et al. 2001). The abilityof ESCs to be continually expanded and contribute tothe formation of the embryo proper are critical charac-teristics that enable complex genetic manipulations inproducing animals for biomedical and agricultural purposes.

However, producing ESCs capable of contributing tothe germline has proven to be challenging in mostspecies.

Limitations in producing chimeras from ESCs have ledto determination of two types of ESCs: naı̈ve and primed(Ying et al. 2008). Naı̈ve (LIF dependent)mESCs possessa high propensity to form chimeric animals, have a high-level clongenicity and demonstrate key differences inepigenetics and gene expression from primed (FGFdependent) mESCs (Nichols and Smith 2009). PrimedmESCs are similar in phenotype and cell signallingregulation to FGF-dependent hESC lines, which aredriving the desire to derive naı̈ve ESCs in many speciesincluding humans (Lengner et al. 2010). The hypothesis isthat naı̈ve state cells will generate a higher proportion ofgermline-competent pluripotent cells (Fig. 1).

Isolation of Embryonic Stem Cells fromNon-Primate and Rodent Species

The development of pluripotent cultures have proven tobe challenging outside of primates and rodents. Whilethere has been progression in multiple species such assheep (Sanna et al. 2010; Bao et al. 2011), swine (Strojeket al. 1990; Vassiliev et al. 2010), cattle (Stice et al. 1996;Cibelli et al. 1998; Saito et al. 2003), horses (Saito et al.2002, 2006; Li et al. 2006), dog (Hatoya et al. 2006;Hayes et al. 2008), cats (Yu et al. 2008; Gomez et al.2010), rabbits (Schoonjans et al. 1996; Wang et al.2007), hamsters (Doetschman et al. 1988), mink(Sukoyan et al. 1993) and chickens (Pain et al. 1996;Horiuchi et al. 2004; van de Lavoir et al. 2006a,b), theyall fail to meet the hallmarks of mESCs. Cells isolatedfrom the ICM of various species often lack evidence ofchimeric formation ⁄germline transmission, which limitstheir use in creating biomedical models and generatingxenotherapeutic tissues. Many factors inhibit the devel-opment of chimeric-competent stem cell from non-primate and rodent species, including isolation andculture techniques. It is hoped that further understand-ing of pluripotency may aid in the development ofchimeric-competent ESCs from these elusive species.

Induced Pluripotent Stem Cells CoreTranscriptional Factors

Through transcriptional profiling, factors involved inthe maintenance of pluripotency have been elucidatedwith Lin28, Pou5f1, Sox 2, Nanog and Nodal beingactively transcribed in all ESCs and additionallyKlf2 ⁄4 ⁄5 and C-myc being important in mESC(Cai et al. 2010). Nanog, Sox 2 and Pou5f1 have been

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identified as ‘core’ factors, demonstrating both regionaland temporal expression in pluripotent cells of the ICM(Nichols et al. 1998; Avilion et al. 2003; Hart et al.2004). These ‘core’ factors are kept in a careful balancethrough positive autoregulation and synergistic regula-tion of other critical pluripotency factors (Boyer et al.2005). Additionally, Nanog has been identified as thekey factor regulating the establishment of the pluripo-tent epigenome (Silva et al. 2006) (Fig. 1). Understand-ing of the role and existence of these core pluripotencyfactors was pivotal in developing cellular reprogram-ming via the overexpression of transcription factors.

Generation of a pluripotent state was only possible inthe case of cell fusion and somatic cell nuclear transfer(McLaren 2000; Do and Scholer 2004) until discovery ofinduced pluripotency by Yamanaka (Takahashi andYamanaka 2006). Starting with 24 prospective genesknown to play a role in pluripotency maintenance, fourfactors (Pou5f1, Sox2, Klf4 and C-myc) were capable ofinitiating an ESC-like state when transduced intosomatic cells (Takahashi and Yamanaka 2006). Usingthe same approach in human fibroblasts, POU5F1,SOX2, KLF4 and C-MYC (Takahashi et al. 2007), orSOX2, POU5F1, NANOG and LIN28 (Yu et al. 2007)also produced iPSCs. Further, experimentation withcellular reprogramming has demonstrated that the onlyindispensible factors are POU5F1 and SOX2 (Eminliet al. 2008; Huangfu et al. 2008; Kim et al. 2008; Parket al. 2008), but the use of NANOG, LIN28, C-MYCand KLF-4 greatly increased reprogramming efficiency.

Although Nanog has been found to be dispensable forthe initiation of reprogramming, it is indispensible for apluripotent state and in its absence, cells can only bepartially reprogrammed (Takahashi and Yamanaka2006; Sridharan et al. 2009). Selecting iPSCs usingNanog expression has enabled more efficient chimerismand increases the incident of germline transmission(Okita et al. 2007). This requirement for Nanog furtherunderscores this gene as a ‘core’ member of pluripotencyfactors, being a gateway to the pluripotent ground state(Silva et al. 2009; Theunissen et al. 2011).

Induced Pluripotent Stem Cell in LivestockSpecies

Recent advances in iPSCs may overcome the roadblocksto establishing pluripotent lines in previously unobtain-able species and demonstrate that the factors of pluri-

potency can cross phylogenic lines. We have shown thathuman factors can be used on pig mesenchymal stemcells (MSCs) to generate iPSCs capable of generatingchimeras with germline transmission (West et al. 2011).These same human pluripotent factors can also formiPSCs from avian species, demonstrating that thesefactors are evolutionarily conserved (Lu et al. 2012).The list of livestock species that show the minimal levelof pluripotency (in vitro and teratoma formation)continues to rapidly increase and now includes sheep(Bao et al. 2011; Li et al. 2011), pigs (Esteban et al.2009; Ezashi et al. 2009; Wu et al. 2009; West et al.2010), rabbits (Honda et al. 2010) and horses (Nagyet al. 2011). Although these discoveries provide hope forthe future with these species in regenerative medicine,most studies have either not tested or reported successfulchimerism. In the pig and quail, we have generatedchimeras using iPSCs reprogrammed using all sixreprogramming factors [POU5F1, NANOG, LIN28,SOX2, KLF4 and C-MYC (PNLSKC)]; the system usedto generate these cells and characteristics is discussedbelow.

Porcine iPSC Generated Using Lentiviral-Based Overexpression of POU5F1, NANOG,LIN28, SOX2, KLF4 and C-MYC

In our porcine studies, we use the lentiviral humanreprogramming factors viPS� kit to overexpress thereprogramming factors PNLSKC driven by an EF1apromoter (West et al. 2010, 2011) to routinely establishmultiple independent stable porcine iPSC (piPSC) lines.These cells consistently demonstrate pluripotency char-acteristics with in vitro and in vivo tests. Our piPSCsdemonstrated, for the first time, that piPSC were capableof contributing to chimeric offspring (West et al. 2010,2011). After initial reprogramming of porcine MSCs,early emerging colonies grown on feeder cells did notappear to be fully reprogrammed; however, once piPSCswere transferred to feeder-free conditions, they showedexpression of the introduced human POU5F1 andSOX2 genes, and independent stem markers SSEA4and TRA 1-81. This proliferative and karyotypic stablecell line has been expanded for 100+ passages withoutchange in phenotype and continues to show robust invitro differentiation potential. Ultimately, injection ofpiPSCs into developing porcine embryos led to theproduction of eight foetuses and 29 live offspringpositive for the human POU5F1 (hPOU5F1) and ⁄orNANOG (hNANOG) genes, indicating that piPSCs hadsuccessfully integrated. Examination of foetal tissuesshowed high levels of chimerism with tissues from allthree germ layers including brain, skin, liver, pancreas,stomach, heart, kidney and spleen being positive for thehPOU5F1 gene. Interestingly, placenta tissue waspositive for hPOU5F1, suggesting that these cellscontributed to the extra-embryonic ectoderm. All spe-cies-specific iPSCs should be tested for ability tocontribution to chimeric offspring to be consideredtruly pluripotent, with the exception of humans, becauseof ethical and moral reasons. This validation is needed,given the reports that less pluripotent mouse iPSC(miPSC) lines can be generated, form teratomas,

Fig. 1. Nanog is the gateway to pluripotency (adopted and modifiedfrom Silva et al. 2009)

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differentiate in vitro, but do not contribute to livechimeric offspring when injected into pre-implantationembryos (Nichols and Smith 2009). More recently, weconfirmed that piPSCs were capable of germline chime-rism for the first time in a non-rodent species (West et al.2010). We obtained a live birth of a transgenic piglet thatpossessed genome integration of the hPOU5F1 andhNANOG genes. Germline-competent piPSCs couldcompletely change the paradigm of how transgenicanimals are produced by making it possible to performmultiple gene knockouts and ⁄or knock-ins to producebiomedical pig models or improve economically impor-tant characteristics for production.

Perhaps of even more importance may be the potentialuse of piPSCs to test the efficacy and safety of noveliPSC therapies in allogeneic or autologous large animalpig models with similarities in anatomy and physiologyto humans. Since the advent of miPSCs, much of thediscussion around this technology has focused on usinga patient’s own cells to generate iPSCs for transplanta-tion. However, the scientific community has questionedthe predictive potential of rodent studies with respect tooutcomes in humans. Recently, derivation of photore-ceptors from piPSCs and the subsequent transplant ofthese cells into pigs devoid of rod photoreceptors whichresulted in successful engrafted and showed strong signsof integrating into the eye tissue has been accomplished(Zhou et al. 2011). This study highlights how the pigcould model potential stem cell therapies. Tumorigenic-ity of iPSCs is another critical concern as previousresearch in the mouse showed that iPSC-derived chime-ras possessed large numbers of tumours. In an initial testto determine the tumorigenicity of piPSCs, our researchgroup performed gross and histological examination ofchimeric animals derived from piPSCs at 2, 7 and 9 months,with all animals developing normally and absent oftumours, although tissue samples from the brain, skin,lung, pancreas, liver, heart and kidney were positive forhPOU5F1 DNA (West et al. 2011). The development ofgermline-competent piPSCs that do not producetumours presents an exciting and powerful translationalmodel to study the efficacy and safety of stem celltherapies and perhaps to efficiently produce complextransgenic animals.

Avian iPSC Generated by Overexpressionof Human Reprogramming Factors

Access to pluripotent stem cells in birds would be ofsignificance in developmental biology studies andtransgenic animal research. It has been demonstratedthat the mechanisms by which POU5F1 and NANOGregulate pluripotency and self-renewal are not exclu-sive to mammals (Lavial et al. 2007), indicating auniversal reprogramming process may exist; however,because of the lack of knowledge of reprogrammingfactors in phylogenetically diverse species, the use ofspecies-specific reprogramming factors to derive iPSCsin these animals is unlikely. In a recent study, wegenerated avian iPSCs using the viPS� kit followingthe established protocol from our piPSCs (West et al.2010). Quail embryonic fibroblasts were transducedwith reprogramming factors and formed quail iPSCs

(qiPSCs) that emerged as colonies in feeder co-cultureand eventually were maintained in feeder-free condi-tions. The derived qiPSC showed typical iPSC char-acteristics as seen in other species (Takahashi andYamanaka 2006; West et al. 2010): they displayedhigh nuclear-to-cytoplasm ratio, prominent nucleoliand telomerase activity >11 fold higher than theparent quail fibroblast cell line. qiPSC also stainedpositive for AP, PAS, POU5F1 and SOX2 and couldbe maintained for >50 passages without loss ofphenotype. In vitro differentiation of qiPSCs showedthat these cells were able to form all three germ layersin EBs and form quail–chick chimeras after injectioninto stage X chicken embryo, demonstrating the bonafide pluripotency of these cells (Lu et al. 2012). Thisfinding in qiPSCs is the first report of the widelyconserved nature of the cellular reprogramming pro-cess in birds, suggesting that it may be a universaltool to derive iPSCs from phylogenetically diversespecies.

As a model animal, avian species have a long historyof providing insight into aspects of developmentalbiology and disease. Avian species are relatively smallin size and the embryo can be easily accessed formanipulation enabling cells or tissues to be trans-planted, which is not possible in mammals (Kulesaet al. 2010). Previous publications reported the differ-entiation of chicken embryonic germ cells (EGs) intomultiple lineages, indicating that these cells may beutilized to perform transplantation studies in birds (Wuet al. 2010). Quail iPSCs are easily maintained in feeder-free and chemical-defined culture systems, and form EBsand demonstrate differentiation into all three germlayers: TUJ1 (ectoderm), SOX17 (endoderm) and alphasmooth muscle actin (aSMA, mesoderm). qiPSCs werealso found to be capable of directed neural differenti-ation expressing the neuron makers Hu C ⁄D and MAP2and the astrocyte and oligodendrocyte proteins GFAPand O4, respectively. These data demonstrated efficientspontaneous and directed differentiation of qiPSCs andthe potential opportunity for use in cell transplantationfor the purpose of developmental and regenerative therapyresearch.

Contribution of iPSCs to chimeric animals is con-sidered the most stringent criteria of pluripotency,indicating cells existing in a naı̈ve or ground state. Wehave recently shown that qiPSCs are capable ofcontributing to the live hatch of chicken–quail chime-ras, even after extended culture (Lu et al. 2012). Inprevious reports, chicken pluripotent cells have beenused to generate chimeric birds (van de Lavoir et al.2006a,b). However, maintaining pluripotent cells inprolonged culture has resulted in decreased chimerismand germline transmission (van de Lavoir et al.2006a,b). qiPSCs in our study are also capable ofclonal expansion after genetic modification, which hasnot been demonstrated before in pluripotent avian linesand would facilitate transgenic animal production.Techniques such as homologous recombinant offerunique opportunities to produce transgenic proteins ineggs, with important pharmaceutical use, or to studygene function in developmental processes using anavian model.

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Conclusion

Beyond expression, we believe the extent and durationof reprogramming factors present in the somatic cellsmay have been important in the generating the firstchimeric-competent livestock iPSCs. Not only is expres-sion needed, but expression levels may need to bedemonstrably higher to achieve a fully reprogrammedstate. Stromal tissue-sourced MSCs express some corepluripotent factors such as POU5F1, but at much lowerlevels than pluripotent stem cells. Bovine ESCs expressPOU5F1 protein, REX1 mRNA and SSEA-1 andSSEA-4 markers and are capable of forming EBs invitro and teratomas in vivo, but not chimeras. The samemarkers and genes including REX1 were expressed insheep iPSC and also have not shown chimerism ability(Bao et al. 2011). Although we have not quantifiedexpression levels of exogenous and endogenous corefactors in our chimera forming livestock iPSC, humaniPSC lines generated using the same reprogramming

system and MOI exhibited extremely high expression ofpluripotent markers. REX1 was approximately 1000fold increased in hiPSC lines than the donor fibroblastcells, and NANOG and POU5F1 were both over 5000fold higher. The key to chimerism in livestock speciesmay be achieving the proper ratio and level of allendogenous and exogenous core pluripotent transcrip-tion factors.

Conflicts of interest

S.L.S. is partially employed and has stock ownership in ArunABiomedical who in collaboration with Thermo Sceintific developed theviPS kit mentioned here.

Funding sources

Georgia Research Alliance and the Bill and Melinda Gates Founda-tions.

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Author’s address (for correspondence):YangqingLu, Department of Animal and DairyScience, Regenerative Bioscience Center,University of Georgia, 425 River Road,Athens, GA 30602, USA. E-mail: sstice@uga.edu

76 Y Lu, JL Mumaw, FD West and SL Stice

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