Author’s address (for correspondence): Yangqing Lu, Department of Animal and Dairy Science, Regenerative Bioscience Center, University of Georgia, 425 River Road, Athens, GA 30602, USA. E-mail: email@example.com
Chimeric animals generated from livestock-induced pluripotent stem cells (iPSCs) have opened the door of opportunity to genetically manipulate species for the production of biomedical models, improving traits of agricultural importance and potentially providing a system to test novel iPSC therapies. The potential of pluripotent stem cells in livestock has long been recognized, with many attempts being chronicled to isolate, culture and characterize pluripotent cells from embryos. However, in most cases, livestock stem cells derived from embryonic sources have failed to reach a pluripotent state marked by the inability to form chimeric animals. The in-depth understanding of core pluripotency factors and the realization of how these factors can be harnessed to reprogram adult cells into an induced pluripotent state has changed the paradigm of livestock stem cells. In this review, we will examine the advancements in iPSC technology in mammalian and avian livestock species.
The discovery of mouse embryonic stem cells (mESCs) in combination with new mammalian gene-altering technologies (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). These advances in biomedical research allowed for the formation of mice which modelled complex diseases generating groundbreaking discoveries in the role of the genetics behind these diseases. Genetically engineered mice have come to be indispensible models for finding novel therapeutics and drug treatments, but they are often unsuitable to accurately model human diseases.
The isolation and culture of pluripotent stem cells, cells capable of forming any cell type of the body, from embryos remained an elusive feat until 1981 when two groups successfully developed a co-culture system that was capable of maintaining mESCs in a pluripotent state with feeder cells secreting necessary factors to maintain pluripotency (Evans and Kaufman 1981; Martin 1981). These first mESC lines demonstrated characteristics that have come to define ESCs in all species. ESCs can be infinitely expanded, differentiated into all three germ layers (endoderm, ectoderm and mesoderm) in vitro as embryoid bodies (EBs) and in vivo in the form of teratomas (Evans and Kaufman 1981; Martin 1981), contributed to all tissue types including the germline in chimeric animals (Bradley et al. 1984) and acted as the inner cell mass (ICM) in tetraploid complementation assays (Eggan et al. 2001). The ability of ESCs to be continually expanded and contribute to the formation of the embryo proper are critical characteristics that enable complex genetic manipulations in producing animals for biomedical and agricultural purposes. However, producing ESCs capable of contributing to the germline has proven to be challenging in most species.
Limitations in producing chimeras from ESCs have led to determination of two types of ESCs: naïve and primed (Ying et al. 2008). Naïve (LIF dependent) mESCs possess a high propensity to form chimeric animals, have a high-level clongenicity and demonstrate key differences in epigenetics and gene expression from primed (FGF dependent) mESCs (Nichols and Smith 2009). Primed mESCs are similar in phenotype and cell signalling regulation to FGF-dependent hESC lines, which are driving the desire to derive naïve ESCs in many species including humans (Lengner et al. 2010). The hypothesis is that naïve state cells will generate a higher proportion of germline-competent pluripotent cells (Fig. 1).
Isolation of Embryonic Stem Cells from Non-Primate and Rodent Species
Through transcriptional profiling, factors involved in the maintenance of pluripotency have been elucidated with Lin28, Pou5f1, Sox 2, Nanog and Nodal being actively transcribed in all ESCs and additionally Klf2/4/5 and C-myc being important in mESC (Cai et al. 2010). Nanog, Sox 2 and Pou5f1 have been identified as ‘core’ factors, demonstrating both regional and 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 balance through positive autoregulation and synergistic regulation of other critical pluripotency factors (Boyer et al. 2005). Additionally, Nanog has been identified as the key factor regulating the establishment of the pluripotent epigenome (Silva et al. 2006) (Fig. 1). Understanding of the role and existence of these core pluripotency factors was pivotal in developing cellular reprogramming via the overexpression of transcription factors.
Although Nanog has been found to be dispensable for the initiation of reprogramming, it is indispensible for a pluripotent state and in its absence, cells can only be partially reprogrammed (Takahashi and Yamanaka 2006; Sridharan et al. 2009). Selecting iPSCs using Nanog expression has enabled more efficient chimerism and increases the incident of germline transmission (Okita et al. 2007). This requirement for Nanog further underscores this gene as a ‘core’ member of pluripotency factors, being a gateway to the pluripotent ground state (Silva et al. 2009; Theunissen et al. 2011).
Induced Pluripotent Stem Cell in Livestock Species
Recent advances in iPSCs may overcome the roadblocks to establishing pluripotent lines in previously unobtainable species and demonstrate that the factors of pluripotency can cross phylogenic lines. We have shown that human factors can be used on pig mesenchymal stem cells (MSCs) to generate iPSCs capable of generating chimeras with germline transmission (West et al. 2011). These same human pluripotent factors can also form iPSCs from avian species, demonstrating that these factors are evolutionarily conserved (Lu et al. 2012). The list of livestock species that show the minimal level of 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 (Nagy et al. 2011). Although these discoveries provide hope for the future with these species in regenerative medicine, most studies have either not tested or reported successful chimerism. In the pig and quail, we have generated chimeras using iPSCs reprogrammed using all six reprogramming factors [POU5F1, NANOG, LIN28, SOX2, KLF4 and C-MYC (PNLSKC)]; the system used to generate these cells and characteristics is discussed below.
Porcine iPSC Generated Using Lentiviral-Based Overexpression of POU5F1, NANOG, LIN28, SOX2, KLF4 and C-MYC
In our porcine studies, we use the lentiviral human reprogramming factors viPS™ kit to overexpress the reprogramming factors PNLSKC driven by an EF1α promoter (West et al. 2010, 2011) to routinely establish multiple independent stable porcine iPSC (piPSC) lines. These cells consistently demonstrate pluripotency characteristics with in vitro and in vivo tests. Our piPSCs demonstrated, for the first time, that piPSC were capable of contributing to chimeric offspring (West et al. 2010, 2011). After initial reprogramming of porcine MSCs, early emerging colonies grown on feeder cells did not appear to be fully reprogrammed; however, once piPSCs were transferred to feeder-free conditions, they showed expression of the introduced human POU5F1 and SOX2 genes, and independent stem markers SSEA4 and TRA 1-81. This proliferative and karyotypic stable cell line has been expanded for 100+ passages without change in phenotype and continues to show robust in vitro differentiation potential. Ultimately, injection of piPSCs into developing porcine embryos led to the production of eight foetuses and 29 live offspring positive for the human POU5F1 (hPOU5F1) and/or NANOG (hNANOG) genes, indicating that piPSCs had successfully integrated. Examination of foetal tissues showed high levels of chimerism with tissues from all three germ layers including brain, skin, liver, pancreas, stomach, heart, kidney and spleen being positive for the hPOU5F1 gene. Interestingly, placenta tissue was positive for hPOU5F1, suggesting that these cells contributed to the extra-embryonic ectoderm. All species-specific iPSCs should be tested for ability to contribution to chimeric offspring to be considered truly pluripotent, with the exception of humans, because of ethical and moral reasons. This validation is needed, given the reports that less pluripotent mouse iPSC (miPSC) lines can be generated, form teratomas, differentiate in vitro, but do not contribute to live chimeric offspring when injected into pre-implantation embryos (Nichols and Smith 2009). More recently, we confirmed that piPSCs were capable of germline chimerism for the first time in a non-rodent species (West et al. 2010). We obtained a live birth of a transgenic piglet that possessed genome integration of the hPOU5F1 and hNANOG genes. Germline-competent piPSCs could completely change the paradigm of how transgenic animals are produced by making it possible to perform multiple gene knockouts and/or knock-ins to produce biomedical pig models or improve economically important characteristics for production.
Perhaps of even more importance may be the potential use of piPSCs to test the efficacy and safety of novel iPSC therapies in allogeneic or autologous large animal pig models with similarities in anatomy and physiology to humans. Since the advent of miPSCs, much of the discussion around this technology has focused on using a patient’s own cells to generate iPSCs for transplantation. However, the scientific community has questioned the predictive potential of rodent studies with respect to outcomes in humans. Recently, derivation of photoreceptors from piPSCs and the subsequent transplant of these cells into pigs devoid of rod photoreceptors which resulted in successful engrafted and showed strong signs of integrating into the eye tissue has been accomplished (Zhou et al. 2011). This study highlights how the pig could model potential stem cell therapies. Tumorigenicity of iPSCs is another critical concern as previous research in the mouse showed that iPSC-derived chimeras possessed large numbers of tumours. In an initial test to determine the tumorigenicity of piPSCs, our research group performed gross and histological examination of chimeric animals derived from piPSCs at 2, 7 and 9 months, with all animals developing normally and absent of tumours, although tissue samples from the brain, skin, lung, pancreas, liver, heart and kidney were positive for hPOU5F1 DNA (West et al. 2011). The development of germline-competent piPSCs that do not produce tumours presents an exciting and powerful translational model to study the efficacy and safety of stem cell therapies and perhaps to efficiently produce complex transgenic animals.
Avian iPSC Generated by Overexpression of Human Reprogramming Factors
Access to pluripotent stem cells in birds would be of significance in developmental biology studies and transgenic animal research. It has been demonstrated that the mechanisms by which POU5F1 and NANOG regulate pluripotency and self-renewal are not exclusive to mammals (Lavial et al. 2007), indicating a universal reprogramming process may exist; however, because of the lack of knowledge of reprogramming factors in phylogenetically diverse species, the use of species-specific reprogramming factors to derive iPSCs in these animals is unlikely. In a recent study, we generated avian iPSCs using the viPS™ kit following the established protocol from our piPSCs (West et al. 2010). Quail embryonic fibroblasts were transduced with reprogramming factors and formed quail iPSCs (qiPSCs) that emerged as colonies in feeder co-culture and eventually were maintained in feeder-free conditions. The derived qiPSC showed typical iPSC characteristics as seen in other species (Takahashi and Yamanaka 2006; West et al. 2010): they displayed high nuclear-to-cytoplasm ratio, prominent nucleoli and telomerase activity >11 fold higher than the parent quail fibroblast cell line. qiPSC also stained positive for AP, PAS, POU5F1 and SOX2 and could be maintained for >50 passages without loss of phenotype. In vitro differentiation of qiPSCs showed that these cells were able to form all three germ layers in EBs and form quail–chick chimeras after injection into stage X chicken embryo, demonstrating the bona fide pluripotency of these cells (Lu et al. 2012). This finding in qiPSCs is the first report of the widely conserved nature of the cellular reprogramming process in birds, suggesting that it may be a universal tool to derive iPSCs from phylogenetically diverse species.
As a model animal, avian species have a long history of providing insight into aspects of developmental biology and disease. Avian species are relatively small in size and the embryo can be easily accessed for manipulation enabling cells or tissues to be transplanted, which is not possible in mammals (Kulesa et al. 2010). Previous publications reported the differentiation of chicken embryonic germ cells (EGs) into multiple lineages, indicating that these cells may be utilized to perform transplantation studies in birds (Wu et al. 2010). Quail iPSCs are easily maintained in feeder-free and chemical-defined culture systems, and form EBs and demonstrate differentiation into all three germ layers: TUJ1 (ectoderm), SOX17 (endoderm) and alpha smooth muscle actin (αSMA, mesoderm). qiPSCs were also found to be capable of directed neural differentiation expressing the neuron makers Hu C/D and MAP2 and the astrocyte and oligodendrocyte proteins GFAP and O4, respectively. These data demonstrated efficient spontaneous and directed differentiation of qiPSCs and the potential opportunity for use in cell transplantation for the purpose of developmental and regenerative therapy research.
Contribution of iPSCs to chimeric animals is considered the most stringent criteria of pluripotency, indicating cells existing in a naïve or ground state. We have recently shown that qiPSCs are capable of contributing to the live hatch of chicken–quail chimeras, even after extended culture (Lu et al. 2012). In previous reports, chicken pluripotent cells have been used to generate chimeric birds (van de Lavoir et al. 2006a,b). However, maintaining pluripotent cells in prolonged culture has resulted in decreased chimerism and germline transmission (van de Lavoir et al. 2006a,b). qiPSCs in our study are also capable of clonal expansion after genetic modification, which has not been demonstrated before in pluripotent avian lines and would facilitate transgenic animal production. Techniques such as homologous recombinant offer unique opportunities to produce transgenic proteins in eggs, with important pharmaceutical use, or to study gene function in developmental processes using an avian model.
Beyond expression, we believe the extent and duration of reprogramming factors present in the somatic cells may have been important in the generating the first chimeric-competent livestock iPSCs. Not only is expression needed, but expression levels may need to be demonstrably higher to achieve a fully reprogrammed state. Stromal tissue-sourced MSCs express some core pluripotent factors such as POU5F1, but at much lower levels than pluripotent stem cells. Bovine ESCs express POU5F1 protein, REX1 mRNA and SSEA-1 and SSEA-4 markers and are capable of forming EBs in vitro and teratomas in vivo, but not chimeras. The same markers and genes including REX1 were expressed in sheep iPSC and also have not shown chimerism ability (Bao et al. 2011). Although we have not quantified expression levels of exogenous and endogenous core factors in our chimera forming livestock iPSC, human iPSC lines generated using the same reprogramming system and MOI exhibited extremely high expression of pluripotent markers. REX1 was approximately 1000 fold increased in hiPSC lines than the donor fibroblast cells, and NANOG and POU5F1 were both over 5000 fold higher. The key to chimerism in livestock species may be achieving the proper ratio and level of all endogenous and exogenous core pluripotent transcription factors.
Conflicts of interest
S.L.S. is partially employed and has stock ownership in ArunA Biomedical who in collaboration with Thermo Sceintific developed the viPS kit mentioned here.
Georgia Research Alliance and the Bill and Melinda Gates Foundations.