Large Animal Models for the Clinical Application of Human Induced Pluripotent Stem Cells

Abstract

Induced pluripotent stem cell (iPSC) technology offers a practically infinite and ethically acceptable source to obtain a variety of somatic cells. Coupled with the biotechnologies of cell therapy or tissue engineering, iPSC technology will enormously contribute to human regenerative medicine. Before clinical application, such human iPSC (hiPSC)-based therapies should be assessed using large animal models that more closely match biological or biomechanical properties of human patients. Therefore, it is critical to generate large animal iPSCs, obtain their iPSC-derived somatic cells, and preclinically evaluate their therapeutic efficacy and safety in large animals. During the past decade, the establishment of iPSC lines of a series of large animal species has been documented, and the acquisition and preclinical evaluation of iPSC-derived somatic cells has also been reported. Despite this progress, significant obstacles, such as obtaining or preserving the bona fide pluripotency of large animal iPSCs, have been encountered. Simultaneously, studies of large animal iPSCs have been overlooked in comparison with those of mouse and hiPSCs, and this field deserves more attention and support due to its important preclinical relevance. Herein, this review will focus on the large animal models of pigs, dogs, horses, and sheep/goats, and summarize current progress, challenges, and potential future directions of research on large animal iPSCs.

Induced Pluripotent Stem Cells and Their Potential Application in Clinic

For the development of regenerative medicine, a source for obtaining unlimited, functional somatic cell types is essential. Since 2006, the emergence of induced pluripotent stem cell (iPSC) technology has fundamentally transformed the biomedical research fields [1]. Early studies showed that murine or human somatic cells could be reprogrammed into a pluripotent stage through ectopic expression of pluripotency-associated transcription factors [1,2]. These iPSCs highly resemble the embryonic stem cells (ESCs) derived from preimplantation blastocysts, particularly in the aspects of robust self-renewability and differentiation capacity into the three germ layer cell derivatives. Additionally, the iPSC technology gained further attractiveness and utility due to its high technical repeatability and reproducibility, high cellular reprogramming efficiency, low economic costs and lack of ethical issues. Hence, the human iPSCs (hiPSCs) virtually provide an infinite reservoir for deriving functional somatic cells for regenerative medicine and associated biomedical research.

During the past decade, the platforms for derivation, maintenance, and differentiation of hiPSCs have been remarkably upgraded to meet the requirements of clinical applications. To date, safe and efficient methods for hiPSC reprogramming and pluripotency maintenance under current good manufacturing practice-compliant conditions, including nonintegrative episomal plasmid-mediated cellular reprogramming and xenogeneic-free culture conditions, have been established and commercialized [3], which has paved the way for the clinical application of hiPSC-based therapies. Simultaneously, these hiPSCs could be efficiently differentiated into many somatic cell types, such as cardiomyocytes, vascular endothelial cells (ECs), and smooth muscle cells, which could even be derived under chemically defined, serum-free conditions [4 –6]. Moreover, the prototypes of a series of hiPSC-based therapies have been established, based on xenogeneically implanting hiPSC cell derivatives into animal models. For example, Dr. Charles Murry's team utilized precryopreserved hiPSC-derived cardiomyocytes to remuscularize substantial amounts of infarcted myocardium of macaque monkeys, and largely remuscularized the ischemic myocardium and reinstalled the cardiac functions [7]. Also, “off-the-shelf” hiPSC-derived Natural Killer cells with novel chimeric antigen receptors were recently generated to specifically target cancer cells in an ovarian cancer mouse xenograft model [8]. Furthermore, hiPSC-derived somatic cells coupled with biocompatible scaffolds have been utilized to fabricate engineered tissues or organs. To date, hiPSC-based, engineered tissues, including cardiac tissue, bone, skeletal muscle, and vascular tissue or grafts have been reported [9 –13]. With the rapid advancement of these technologies, hiPSCs may offer promising opportunities for clinical treatment of a spectrum of diseases.

Large Animal Models for Preclinical Evaluation of iPSC-Based Therapies

Before clinical application, animal models, which simulate human biology, should be utilized to assess the safety and efficacy of such iPSC-based treatments. These preclinical animal studies are expected to lead to reproducible outcomes and conclusive knowledge or experience, which can eventually guide the clinical application of the evaluated therapies. Currently, rodent species remain the most widely used animal model in the entire biomedical research field. Due to their short lifespans and reproductive cycles, small body sizes, accessibility to a comprehensively sequenced genome, and genetically manipulatable murine ESCs, rodent species are easy to operate and allow scientists to effectively uncover gene functions and mechanisms of pathological disorders [14]. However, the genetics, pathology, and physiology of rodents display prominent evolutionary deviations from those of humans. For instance, the mouse model of atherosclerosis cannot be naturally established unless the metabolism of cholesterol is genetically modified, since mice lack the cholesteryl ester transfer protein, present causative lower plasma cholesterol levels, and are resistant to the formation of atherosclerosis [15]. Another example is that of human patients with genetic deficiency of phospholamban, an intracellular calcium regulator in cardiomyocytes, who present severe dilated cardiomyopathy and required cardiac transplantation [16], whereas phospholamban knockout mice in contrast show enhanced cardiac contraction without risk of mortality [17]. Hence, murine models typically do not recapitulate all the characteristics of the natural human phenotype, and may even present opposite phenotypes, which makes them of lowered utility as a model for preclinical, translational studies.

An ideal mammalian species as a preclinical model should display the natural existence of biological, physiological, and pathological conditions comparable to those of human, an adequate capacity for long-term handling, experiments, operations and maintenance, minimal ethical concerns, and minimal financial cost. Although nonhuman primate models highly simulate human biology, the heavy financial burden and sensitive ethical issues make them less practical, and hamper their widespread application in research [18]. Alternatively, domestic large animals, such as pigs, dogs, horses, goats, and sheep, could be imperative models in preclinical research due to their comparable biological properties to human, easy accessibility, and lowered ethical controversy. To mimic and evaluate hiPSC-based therapies, large animal iPSCs should be generated through comparable techniques for deriving clinical-grade hiPSCs, and the cell or tissue materials obtained from these iPSCs should be further utilized to treat the intended animals (Fig. 1).

FIG. 1.

Application of large animal model (A) in preclinical evaluation of iPSC-based therapy for human regenerative medicine (B).

To date, a considerable amount of studies on large animal iPSCs have been accomplished, and the publications on iPSCs of several major species are visualized in Fig. 2. In general, the field of large animal iPSCs deserves significant attention and has undergone remarkable progress, but remains overlooked when compared with the enormous amount of publications and rapid development of rodent and hiPSC studies. The majority of these large animal studies focused on derivation of iPSC lines and understanding the mechanism of cellular reprogramming or pluripotency regulation, while a portion of studies described iPSC differentiation toward specific cell types. Among the large animal modeling field, pig iPSCs appear the most advanced with a considerably larger number of publications relative to other species. This review will focus on the iPSCs of pigs, dogs, horses, and sheep/goats, and discuss their current progress, potential challenges, and future directions.

FIG. 2.

Illustrative summary of publications on large animal iPSCs to date. The number of publications on iPSCs of pig, dog, horse, and goats/sheep are listed as each column. For each species, the publications are divided into four categories based on the research perspectives starting at the bottom of the y-axis and moving up: (i) iPSC line establishment and characterization; (ii) mechanism of cellular reprogramming or pluripotency maintenance; (iii) Directed differentiation of iPSCs into somatic cell types; (iv) Application of iPSC-derived somatic cells in tissue engineering or preclinical treatment.

Porcine iPSCs

Advantages of porcine models

Since pigs and humans display a high degree of similarity in many biological perspectives, affordable cost for maintenance, and minimal ethical issues in comparison to many other animal models, enormous biomedical studies have been reported using pig models to date [19,20]. For instance, due to the remarkable similarities of porcine heart-to-body weight ratio, cardiac size, coronary artery anatomy distribution, cardiovascular physiology, cardiomyocyte metabolism, cardiac electrophysiology, and the pathological response to myocardial ischemia to those of human, the pig model is widely utilized in cardiovascular studies, especially for those associated with myocardial infarction and coronary artery diseases [21,22]. Porcine models were also applied in several regenerative medicine studies to assess hiPSC-derived cardiomyocyte-based treatment for heart repair [23,24]. Among the modern porcine breeds, the Yorkshire pigs have been widely accepted as a reliable preclinical animal model in biomedical research. However, adult Yorkshire pigs can reach a body weight of 249–306 kg, which is challenging for husbandry and handling in biomedical studies [14]. In comparison, the miniature porcine breeds, such as Yucatan and Göttingen, are receiving increased attention, as their body weight will reach 68–91 kg at maturity, approaching the average body weight of human adult males [14].

The development of an inbred pig platform could further broaden the scope of application of porcine model. To mimic autologous hiPSC-based therapy, large animal iPSC lines should be generated from the somatic cells of each individual pig, differentiated, and transplanted back to the original pig donor. Such an experimental approach could be laborious, lack scalability, and potentially lead to results with high variability between individual animals. In contrast, intensive inbreeding can ultimately reduce heterozygosity and increase immunocompatibility between individual animals. Therefore, the inbred porcine model would allow the implantation of cellular or tissue products derived from one single porcine iPSC (piPSC) line into multiple inbred pig recipients, which would tremendously scale up experiments, reducing the costs and elevating the efficiency. Dr. David Sachs's group from Massachusetts General Hospital (MGH) established the MGH inbred miniature pig model with a growing body of associated publications. The MGH pigs with long-term inbreeding were reported to accept skin graft of siblings for greater than 340 days and hearts for greater than 265 days [25]. So far, somatic cells from MGH inbred pigs have been utilized to generate inbred piPSC lines independently by several laboratories [26,27].

Progress in piPSCs

piPSCs are currently the most developed field among those of all large animal iPSCs with 85 publications (Fig. 2). piPSCs were initially generated using retrovirus or lentivirus-mediated reprogramming methods to deliver the human or mouse pluripotency-associated transcription factors into porcine embryonic fibroblasts, respectively [28,29]. Subsequently, a growing body of studies applied various reprogramming approaches to obtain piPSCs, including the doxycycline-inducible reprogramming system [27,30,31], piggyBac transposon [32], episomal plasmid [33], and Sendai virus [34,35]. Besides using the Yamanaka factors (OCT4, SOX2, KLF4, and c-MYC), other pluripotency-associated transcription factors, such as NANOG and LIN-28, were also utilized to enhance the reprogramming efficiency [26,34]. Moreover, porcine embryonic fibroblasts were the most commonly chosen donor cell type for piPSC generation, while other somatic cell types, including porcine adipose-derived stromal cells and porcine dermal fibroblasts were occasionally utilized [26,36].

To maintain the pluripotency, piPSCs were regularly expanded in the presence of feeder cells such as mouse embryonic fibroblasts, whereas piPSCs cultured on matrigel-based, feeder-free condition have also been reported [37]. The leukemia inhibitory factor (LIF) [38], or the basic fibroblast growth factor (bFGF) [28], or both LIF and bFGF [27] have been provided in the culture medium to support the pluripotency of piPSCs. The piPSCs, in general, presented representative features of pluripotency, including the bioactivity of alkaline phosphatase, expression of endogenous pluripotency markers such as porcine OCT4, SOX2, and NANOG, the capability of differentiation toward three germ layer cell derivatives through in vitro embryoid body formation and in vivo teratogenesis. However, the expression profiles of pluripotency-associated surface markers in piPSCs, such as SSEA1, SSEA3, SSEA4, TRA-1-60, and TRA-1-81, dramatically deviated between different reports [28,29]. The divergence in growth factor requirements and marker expression profiles indicated that the nature of pluripotency of these piPSCs might display an inclination to that of either a naive inner cell mass or primed epiblast stem cells [39]. Furthermore, small molecules as inhibitors of certain signaling pathways have been applied to piPSC studies. Zhang et al. suggested that the piPSCs at a naive pluripotency stage could be achieved by inhibiting ERK1/2 and GS3K pathways in the presence of LIF [40]. However, it was also described previously that the inhibition of ERK1/2 and GS3K pathways reduces expression of pluripotency-related genes [41]. It is noteworthy that Ma et al. described that through a novel combination of small-molecule inhibitors, including the inhibitors of TGF beta-SMAD2/3, ERK1/2, and GS3K pathways, the pluripotency of doxycycline-inducible piPSCs could be escalated when ectopic genes were inactivated by the withdrawal of doxycycline [42].

piPSCs have been differentiated into a variety of functional somatic cell types. To date, the differentiation of piPSCs toward hepatocytes [43,44], neural cells [35,45 –48], skeletal myocytes [49], ECs[36], vascular smooth muscle cells (VSMCs) [27], and rod photoreceptors [50] has been accomplished. Zhou et al. successfully attained photoreceptor differentiation using piPSCs [50]. These piPSC-derived rod photoreceptors presented a series of photoreceptor markers and were subsequently transplanted into the subretinal space of pigs previously treated with iodoacetic acid to eliminate the local primary rod photoreceptors. The engrafted piPSC-derived rod photoreceptors presented evident signs of integration into the surrounding tissue. Considering the high anatomical and physiological similarities between porcine and human eyes, this study could provide important information for using the pig model to simulate hiPSC-derived retinal stem cell transplantation. Moreover, successful derivation of ECs from piPSCs has been reported by Gu et al. [36]. In this study, piPSC-derived ECs presented comparable morphology and functions as porcine primary aortic ECs. The therapeutic potential of piPSC-derived ECs was assessed by grafting these cells into ischemic myocardial tissue of mice. In contrast to the control group, mice with engrafted piPSC-derived ECs displayed signs of improved cardiac function as indicated by results of echocardiography and magnetic resonance imaging. Further mechanistic study suggested that the piPSC-derived ECs secreted proangiogenic and antiapoptotic factors in ischemic cardiac tissue, and causatively promoted neovascularization and cardiomyocyte survival. Additionally, a recent study by Strnadel et al. reported that the syngeneic piPSC-derived neural precursor cells (NPCs) were efficiently derived and transplanted to the porcine spinal cord without immunosuppression [35]. The grafted piPSC-NPCs demonstrated long-term survival with neuronal and glial differentiation without significant signs of immunorejection, which largely eliminates the concern of unexpected immune rejection of undifferentiated autologous mouse iPSCs after transplantation [51]. This study strongly supported that hiPSC could be a potential therapeutic source of deriving transplantable NPCs for treating neural disorders in the spinal cord. Moreover, in the perspective of tissue engineering-based regenerative medicine, functional VSMCs have been successfully derived from inbred piPSCs, and were utilized to generate the vascular tissues by self-assembly or growing on biodegradable scaffolds used in constructing tissue-engineered vascular grafts, which suggested the potential of piPSC-derived VSMCs in developing vascular grafts [27]. It can be envisioned that such piPSC-based vascular grafts could be generated from one single inbred piPSC line, and implanted into multiple inbred pig siblings for a scaled-up evaluation of the efficacy and safety of these iPSC-based vascular grafts [12].

Challenges in piPSCs

In contrast to human or murine iPSCs, the pluripotency of piPSCs appears to require constant expression of ectopic pluripotency genes, which has been one of the central issues preventing the progress of this field. Constant expression of ectopic pluripotency genes in piPSCs may prevent the efficient differentiation toward desired somatic cell types, or trigger the unwanted formation of teratoma after engraftment into a pig recipient, which will consequently disturb the preclinical evaluation of the efficacy and safety of an iPSC-based therapy. As a result, dramatic amounts of effort have been put into addressing this issue, and 43 publications out of the total 85 publications associated to piPSC studies to date (50.59%) focus on the mechanism of reprogramming and pluripotency regulation (Fig. 2).

Ezashi et al. and Esteban et al. initially and independently reported that the expandable piPSC clones generated through a retrovirus-mediated method presented constant expression of ectopic human pluripotency genes without being silenced [28,29]. West et al. uncovered that piPSCs could contribute to the generation of germline chimeras, whereas the chimera tissues were strikingly positive for ectopic human OCT4 expression [52]. Moreover, studies using piPSCs with doxycycline-inducible pluripotency suggested that the inactivation of ectopic pluripotency genes by removal of doxycycline caused immediate loss of pluripotency [27,53]. These results suggest that ectopic pluripotency genes could not improve the autonomy of the endogenous pluripotency genes of piPSCs [28,54]. It is noteworthy that the nonintegrative episomal plasmids have been applied to generate piPSCs, but these piPSCs displayed persistent expression of exogenous OCT4, as a consequence of unexpected genomic integration of the episomal plasmid [33]. In contrast, nonintegrative Sendai virus-mediated reprogramming approaches have been utilized to generate the piPSCs [35]. Neuronal cells were derived from these piPSCs and successfully grafted into the rat striata or swine spinal cord for 3 or 7 months, respectively. The graft sections displayed the absence of OCT4 or KLF4 indicated by immunostaining assay. Although this result was promising, the study lacked examination of genomic integration of ectopic transgenes in these piPSCs. Hence, it remains possible that the pluripotency genes delivered by Sendai virus were accidently inserted into the genome, and the expression of these genes were gradually silenced along with differentiation and engraftment. The pluripotency-associated regulatory mechanisms in porcine ESCs (pESCs) should provide clues of the regulation of porcine pluripotency. However, the long-term maintenance of pluripotent pESCs has proved challenging without overexpression of ectopic OCT4 in pESCs [55].

It has been gradually recognized that epigenetic barriers may exist within piPSCs under the current reprogramming and culture conditions, which consequently prevent the expression of endogenous pluripotency genes. Choi et al. evaluated the levels of DNA methylation at the promoter regions of several porcine endogenous pluripotency genes in piPSCs, and results revealed that the promoter of porcine OCT4, but not NANOG, remained heavily methylated in piPSCs in comparison with that of porcine fibroblasts [53]. Du et al. reported that the porcine OCT4 promoter regions in piPSCs were comparably methylated as those in porcine fibroblasts, whereas the OCT4 promoter regions of the inner cell mass of porcine blastocysts were completely unmethylated [56]. The epigenetic modifications to histones in piPSCs has also been documented. Utilizing the piPSCs previously generated by West et al. [57], Xiao et al. have accomplished a comprehensive analysis of the epigenetic profile, such as the genomic distributions of cytosine methylation, H2A.Z, H3K4me1/2/3, H3K9me3, H3K27me3, H3K27ac, and H3K36me3 [58]. Moreover, Xie et al. explored that levels of the repressive epigenetic marker H3K27me3 in porcine cloned embryos were significantly higher than that of in vitro fertilized pig embryos, and that efficiency of piPSC generation could be elevated when reducing the H3K27me3 levels in donor cells [59]. In contrast, Wu et al. reported that only minimal repressive epigenetic modifications existed in doxycycline-inducible piPSCs [31].

As such, efforts have been made to overcome the epigenetic barriers in piPSCs. Recently, Mao et al. suggested that piPSCs could resemble the “F-class” iPSCs, which are typified by NANOG positiveness, the dependence on high expression of reprogramming factors, and the low expression levels of core endogenous pluripotency genes [60]. The authors further hypothesized that these F-class iPSCs could be converted to transgene-independent, ESC-like cells if treated with small-molecule epigenetic modifier inhibitors, such as histone deacetylase inhibitors (HDACi), sodium butyrate (NaB), or trichostatin A (TSA). Driven by this hypothesis, piPSCs were generated through retrovirus-mediated ectopic expression of pluripotency genes in combination with the epigenetic modifier genes, including Ten-Eleven Translocation (TET1 or TET3) or lysine-specific demethylase 3A, and these piPSCs expressed elevated levels of essential genes representing a naive state and exhibited more open chromatin status compared with piPSCs derived with ectopic expression of pluripotency genes only. Furthermore, if coupled with the addition of HDACi, NaB, TSA, or valproic acid, the expression of representative genes of a naive pluripotent state can be further increased and, interestingly, the expression of exogenous pluripotency genes decreased. This study conspicuously pointed out that the manipulation of epigenetic modification in piPSCs may lead to an independence from ectopic pluripotency genes. This study could also be limited by the potential interference of constant expression of ectopic transgenes in piPSCs, which might simultaneously interfere with the activity of an epigenetic modifier. In the future, piPSC lines with doxycycline-inducible pluripotency could be used to select the epigenetic modifier genes or small-molecule drugs. The effects of gene or drug candidates should be ultimately evaluated by generating the piPSCs through nonintegrative reprogramming methods such as the Sendai virus. Additionally, it has been archived that the effective demethylation of DNA and H3K9 at the OCT4 promoter by using 3-Deazaneplanocin A hydrochloride (DZNep), a histone methyltransferase EZH2 inhibitor, played an essential role in the successful reprogramming of mouse somatic cells to pluripotency using only small-molecule compounds [61]. It was indicated that DZNep might play a positive role in enhancing the reprogramming efficiency through porcine somatic cell nuclear transfer (SCNT) [62]. Moreover, recent success in nonhuman primate cloning revealed the significant contribution of TSA treatment and the transient enhancement of Kdm4d expression, a H3K9me3 demethylase, into the cloned embryos [63], which may eventually assist the generation of piPSCs with true pluripotency.

In a very recent report, Gao et al. utilized the OCT4–tdTomato⁺, doxycycline-inducible piPSCs and drug screening system, and defined the medium ingredients that maintained the pluripotency in both piPSCs and pESCs [64]. With the presence of LIF, activin A and a series of small molecules to modulate the ERK, WNT, and SRC pathways in culture medium, the piPSCs and pESCs were expanded and demonstrated the capacity of pluripotency, revealed by the results of chimera formation assay and a number of other classic pluripotency characterizations. This achievement is encouraging and largely push the application of piPSC model for preclinical use forward.

Canine iPSCs

Advantages of canine models

The experience and knowledge of over five decades from preclinical canine models has profoundly enhanced our understanding of human clinical diseases and corresponding therapeutic avenues. Dogs have larger body sizes and longer lifespans than rodents, and have similar relative organ positions and present biochemical and pathological similarities to humans [65]. To date, research in dogs has provided fundamental knowledge for bone marrow transplantation, metabolic diseases, neurological disorders, cancers, and heart failure [65 –69]. Moreover, the modern canine breeds have displayed significant variety in size, appearance, and behavior, which is the consequence of long-term breeding with human intervention. Canine genetic variation offers a natural model for analyzing genetic disorders. Over 400 types of canine genetic disorders have been identified, and about half of these canine diseases resemble those in humans, including cardiomyopathies and muscular dystrophy [70,71]. In addition, due to the variant anatomical features of different canine breeds, dogs may provide unique models for certain human diseases or injuries. One example is the high prevalence of spinal cord injury (SCI) and causative paralysis in breeds with long body length but short limbs such as Dachshunds or Beagles. Approximately 77% of dogs suffering from SCI present intervertebral disc degeneration [72 –75]. The application of stem cells to treat pathological conditions in the dog that would ordinarily lead to disability or impact on life quality, would benefit the dogs as popular companion animals, but also will tremendously promote the progress of human regenerative medicine. The generation and application of canine iPSCs (ciPSCs) may allow the evaluation of the safety and efficacy of hiPSC-based therapy.

Canine iPSCs

Although the topics of published studies on ciPSCs covered the generation, differentiation, and preclinical application of the ciPSCs, this field was generally less developed than that of piPSCs, with a total of 15 published studies up to now, with about half of the publications focusing on the primitive stages of generating ciPSC lines (Fig. 2).

The first brief report of ciPSC generation was published in 2010 by Shimada et al. [76], and two more groups immediately published their success in establishing ciPSC lines with more comprehensive characterization in the next year [77,78]. Similar to canine ESCs and human pluripotent stem cells, ciPSCs display pluripotency markers, such as OCT4 and NANOG, and differentiation capacity toward the three germ layers. Different from human or mouse pluripotent stem cells, however, ciPSCs required the presence of the dual growth factors LIF and bFGF to sustain the pluripotency [76 –78]. Research by Luo and Cibelli indicated that LIF is specifically critical for survival of ciPSCs, whereas bFGF manages ciPSC's pluripotency through a mechanism highly similar to that of ESCs with primed pluripotency, [79] namely ciPSCs were apoptotic and displayed escalated activation of caspase-3 in the absence of LIF [79]. In comparison, without bFGF, ciPSCs rapidly decreased the expression of NANOG, but not OCT4 or SOX2, and comparable expression alterations in ciPSCs could be observed when the SMAD2/3 pathway was inhibited. Moreover, similar to human ESCs, activin-A could replace bFGF to maintain NANOG expression and pluripotency of ciPSCs [80]. These results suggested that ciPSCs may resemble the epiblast stem cells with “primed” pluripotency. In contrast to the study above, ciPSCs dependent on LIF alone have also been reported [81,82]. These LIF-dependent ciPSCs presented the typical expression profile of pluripotency genes as well as robust lineage differentiation potential. It is worth noting that Whitworth et al. reported that the LIF-dependent ciPSCs displayed trimethylation of histone H3K27, indicating that these ciPSCs resemble inner cell mass-derived cells with naive pluripotency [82]. In the future, more endeavors should be made to understand the key mechanisms of pluripotency regulation in LIF-dependent ciPSCs.

Furthermore, ciPSCs can be specifically differentiated into somatic cell types. The derivation of mature megakaryocytes and platelets from ciPSCs by Nishimura et al. was encouraging [83], since this study may initiate a clinical model for stem cell-based therapy of thrombocytopenia, a blood disorder common to canines and human beings, currently requiring blood transfusion as the only valid therapy. The generation of mesenchymal stromal cells with typical differentiation capability toward adipocytes, osteocytes, and chondrocytes was independently reported by two groups [84,85] and these ciPSC-derived mesenchymal cells may provide a vital source of interstitial cells for regenerative medicine. In addition, Lee et al. demonstrated the therapeutic potential of ciPSC-derived ECs in treating myocardial infarction and hindlimb ischemia [77].

Distinct from the challenging situation of piPSCs, the pluripotency in ciPSCs appears to not be dependent on the constant expression of ectopic pluripotency genes. However, the research of ciPSCs remains in the zone of iPSC line establishment. One reason which has hindered the development of ciPSCs is that biochemical cues for leveraging the pluripotency and viability of ciPSCs remain obscure. Potentially, due to the limitation of current culture conditions, the lack of robust and efficient ciPSC generation[82], and the fact that ciPSCs displayed limited capacity for teratogenesis with immature structures of the three germ layer tissues in several reports [77 –79,81], suggest that the current culture conditions are not sufficient to maintain the complete pluripotency of ciPSCs. Also, inhibition of the LIF-STAT3 pathway could immediately harm the viability of ciPSCs, which indicated that the JAK-STAT3 pathway might be compensating a constant, vital stress from current culture conditions [79]. A comprehensive study should be undertaken to elucidate the trophic requirement for maintaining ciPSCs. It could be promising to take advantage of drug-inducible ciPSCs [86] or ciPSCs with an OCT4-based cellular reporter to select the optimum components for culture conditions, such as the growth factors, serum supplementation, oxygen, pH, and osmotic concentration of the culture medium. Moreover, the utilization of clinical-grade reprogramming methods for ciPSC line establishment is required for preclinical evaluation of stem cell-based therapies. To date, most of the reported ciPSC lines were developed based on retrovirus or lentivirus-mediated reprogramming, which integrates the exogenous genes into canine genome. Furthermore, some studies reported the risk of unwanted expression of integrated ectopic pluripotency genes in ciPSCs [81,84]. A genomic integration-free reprogramming approach should be developed for ciPSC generation to match the clinical requirements. It is noteworthy that recently Chow et al. and Tsukamoto et al. independently generated ciPSCs using commercially available, Sendai virus-mediated reprogramming [85,87], and these cells demonstrated classic pluripotency gene expression profiles and teratogenicity in immunodeficient mice models. With reproducible results based on well-defined culture conditions at a clinical grade, the research of ciPSCs may achieve steady improvement.

Equine and Ovine iPSCs

Other livestock species, such as horses, goats, and sheep, have also been contributing to studies for biomedical sciences, particularly with research on musculoskeletal disorders. Due to the frequent exposure of horses to intense physical activity through sporting events, they could be utilized as a preclinical model of musculoskeletal injuries, such as bone fracture and osteoarthritis, in tendon or skeletal muscle [88]. Similarly, goat and sheep models for osteoporosis research have been established with a considerable amount of publications [89]. Hence, it is theoretically reasonable to evaluate iPSC-based therapy to regenerate functional musculoskeletal tissues in horses, sheep, and goats. However, confined by the highly specialized application to human medical research, research on horse, sheep, and goat iPSCs has received limited attention, and the research progress has been generally confined to the primitive stages of pluripotency induction and maintenance.

Equine iPSCs (eiPSCs) and their cell derivatives have been generated and described in 17 reports to date (Fig. 2). Equine fibroblasts [90,91], keratinocytes [92], or adipose-derived stem cells [93] were used for eiPSC generation, and eiPSCs could be generated through methods mediated by lentiviral vectors [93,94] or piggyBac transposons [91,95]. In general, the eiPSCs could be stable for long-term expansion above 60 passages [96], and the pluripotency maintenance was dependent on the presence of LIF [92] or both LIF and bFGF [91]. Furthermore, horse iPSCs were reported to be differentiated into a number of somatic cell types, including keratinocytes [97], skeletal muscle cells [94,98,99], mesenchymal-like cells [100], chondrocytes[98,100], osteoblasts, and cortical neurons [101]. A recent publication by Baird et al. reported the generation of a three-dimensional printed, biocompatible, thermoplastic scaffold for efficient osteoblast differentiation of eiPSCs [99].

In comparison, studies on sheep and goat iPSCs are less developed with only eight publications (Fig. 2) and essentially focused on the generation and characterization of sheep or goat iPSCs. Liu et al. successfully generated sheep iPSCs by retrovirus-mediated reprogramming [102]. These sheep iPSCs were maintained in the presence of bFGF and displayed the capacity of pluripotency, teratogenesis, and contribution to inner cell mass formation. Recently, Tai et al. reported that goat iPSCs could be generated using a doxycycline-inducible reprogramming system, and they were maintained at pluripotency status in a medium containing mouse LIF [103]. These goat iPSCs could preserve pluripotency and present teratogenesis when expression of ectopic factors was shut down by withdrawal of doxycycline. Concurrently, German et al. established ovine iPSC lines by retrovirus-mediated reprogramming and expanded them in a medium containing bFGF [104]. These ovine iPSCs provided indications of pluripotency, while exogenous factors appeared to be actively expressing in ovine iPSCs. Moreover, these ovine iPSCs were subjected to SCNT and cloned blastocysts were derived. However, the preimplantation development of iPSC-SCNT embryos was less efficient than with ovine somatic cells, and the exogenous genes remained actively expressing in the cloned blastocysts.

Conclusion and Future Directions

In summary, numerous studies on large animal iPSCs have been archived, and have displayed the great application potential of these iPSCs for biomedical studies. The research on large animal iPSCs deserves significant attention owing to their critical position in the development of iPSC-based regenerative medicine, and the experimental outcomes, experiences, and knowledge derived from large animal iPSC-based preclinical trials will effectively support the clinical application of iPSC-regenerative medicine. However, this field has been generally overlooked during the past decade, especially when compared with the rapid evolution of iPSC technology for rodent species and humans, which is principally due to the technical difficulties in establishing a standardized, clearly defined, efficient, and reproducible culture condition for cellular reprogramming and pluripotency maintenance. This corresponds to the present situation that the majority of studies on large animal iPSCs appear to provide sporadic fragments of knowledge and linger at the levels of pluripotency establishment (Fig. 2). To achieve the breakthrough in this field, more attention should be paid and endeavors undertaken. First, research on blastocyst-derived ESCs should be re-emphasized. Considering that blastocyst-derived ESCs may be highly informative as a positive control of pluripotent stem cells, more effort should be put toward understanding the pluripotency-associated regulatory mechanisms in large animal ESCs, which may provide solid knowledge and insights that benefit the pluripotency maintenance of large animal iPSCs. A very recent report by Choi et al. demonstrated that the combination of a series of critical conditions, including the inhibition of Wnt and GSK3 pathways, the addition of bFGF and activin A and the supplement of defined lipid concentrates, could remarkably enhance the derivation efficiency and the pluripotency maintenance of pESCs [105]. This is the first time that pESC lines could be established effectively and subcultured continuously for 50 passages while maintaining normal karyotypes and pluripotency, which may ultimately further support the exploration of the optimum conditions and the pluripotency-associated regulatory mechanisms for generating piPSCs. Next, an effective culture condition-selecting system, such as using OCT4-based green fluorescent protein reporter iPSC lines or doxycycline-inducible iPSC lines, should be established to screen the conditions for supporting pluripotency of large animal iPSCs. Additionally, the application of novel biochemical and biophysical conditions to facilitate pluripotency maintenance should be considered and encouraged during the generation and culture of large animal iPSCs, such as the utilization of large animal serum or platelet lysate [42], or cell culture on a soft substrate [106,107].

Simultaneously, it is critical for the large animal iPSC model to meet the potential requirements of the latest progress in regenerative medicine. Recent studies have underscored the potential application of hypoimmunogenic hiPSCs, generated by modulating the expression of human major histocompatibility complex (MHC), as an allogeneic but universally immunocompatible, off-the-shelf donor cell source in regenerative medicine [108,109]. The desired somatic cell types derived from such hypoimmunogenic donor iPSCs could be produced and cryopreserved on a large scale in advance and immediately utilized for emergency treatment, which may dramatically increase the potential application of iPSC-based therapy as a readily or promptly available treatment for patients. Therefore, by genetically eliminating the expression of MHC molecules, the hypoimmunogenic iPSCs of large animal species could be established as a preclinical model to evaluate the safety and efficacy of allograft of this universal donor cell-based therapy.

Footnotes

Acknowledgments

Due to space limitations, the authors are not able to recruit all important articles on large animal iPSCs, and they apologize to those investigators whose articles they omitted in this study. The authors also thank Dr. Jose Cibelli (Michigan State University) and Dr. Yibing Qyang (Yale University) for their strong support. The strong and consistent support from Mr. Ling Luo and Mrs. Jie Yang is gratefully appreciated.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

References

Takahashi

and Yamanaka

. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126:663–676.

, Vodyanik

, Smuga-Otto

, Antosiewicz-Bourget

, Frane

, Tian

, Nie

, Jonsdottir

, Ruotti

, et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science, 318:1917–1920.

Baghbaderani

, Tian

, Neo

, Burkall

, Dimezzo

, Sierra

, Zeng

, Warren

, Kovarcik

, Fellner

and Rao

. (2015). cGMP-manufactured human induced pluripotent stem cells are available for pre-clinical and clinical applications. Stem Cell Rep, 5:647–659.

Lian

, Bao

, Zilberter

, Westman

, Fisahn

, Hsiao

, Hazeltine

, Dunn

, Kamp

and Palecek

. (2015). Chemically defined, albumin-free human cardiomyocyte generation. Nat Methods, 12:595–596.

Patsch

, Challet-Meylan

, Thoma

, Urich

, Heckel

, O'Sullivan

, Grainger

, Kapp

, Sun

, et al. (2015). Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells. Nat Cell Biol, 17:994–1003.

Cheung

, Bernardo

, Trotter

, Pedersen

and Sinha

. (2012). Generation of human vascular smooth muscle subtypes provides insight into embryological origin-dependent disease susceptibility. Nat Biotechnol, 30:165–173.

Liu

, Chen

, Yang

, Fugate

, Kalucki

, Futakuchi-Tsuchida

, Couture

, Vogel

, Astley

, et al. (2018). Erratum: human embryonic stem cell-derived cardiomyocytes restore function in infarcted hearts of non-human primates. Nat Biotechnol, 36:899.

, Hermanson

, Moriarity

and Kaufman

. (2018). Human iPSC-derived natural killer cells engineered with chimeric antigen receptors enhance anti-tumor activity. Cell Stem Cell, 23:181–192.e5.

Maffioletti

, Sarcar

, Henderson

ABH

, Mannhardt

, Pinton

, Moyle

, Steele-Stallard

, Cappellari

, Wells

, et al. (2018). Three-dimensional human ipsc-derived artificial skeletal muscles model muscular dystrophies and enable multilineage tissue engineering. Cell Rep, 23:899–908.

10.

de Peppo

, Marcos-Campos

, Kahler

, Alsalman

, Shang

, Vunjak-Novakovic

and Marolt

. (2013). Engineering bone tissue substitutes from human induced pluripotent stem cells. Proc Natl Acad Sci U S A, 110:8680–8685.

11.

Ronaldson-Bouchard

, Ma

, Yeager

, Chen

, Song

, Sirabella

, Morikawa

, Teles

, Yazawa

and Vunjak-Novakovic

. (2018). Advanced maturation of human cardiac tissue grown from pluripotent stem cells. Nature, 556:239–243.

12.

Gui

, Dash

, Luo

, Qin

, Zhao

, Yamamoto

, Hashimoto

, Wu

, Dardik

, et al. (2016). Implantable tissue-engineered blood vessels from human induced pluripotent stem cells. Biomaterials, 102:120–129.

13.

Dash

, Levi

, Schwan

, Luo

, Bartulos

, Wu

, Qiu

, Yi

, Ren

, et al. (2016). Tissue-engineered vascular rings from human iPSC-derived smooth muscle cells. Stem Cell Rep, 7:19–28.

14.

Gutierrez

, Dicks

, Glanzner

, Agellon

and Bordignon

. (2015). Efficacy of the porcine species in biomedical research. Front Genet, 6:293.

15.

Hampton

. (2017). How useful are mouse models for understanding human atherosclerosis? Review examines the available evidence. Circulation, 135:1757–1758.

16.

Haghighi

, Kolokathis

, Pater

, Lynch

, Asahi

, Gramolini

, Fan

, Tsiapras

, Hahn

, et al. (2003). Human phospholamban null results in lethal dilated cardiomyopathy revealing a critical difference between mouse and human. J Clin Invest, 111:869–876.

17.

Slack

, Grupp

, Dash

, Holder

, Schmidt

, Gerst

, Tamura

, Tilgmann

, James

, et al. (2001). The enhanced contractility of the phospholamban-deficient mouse heart persists with aging. J Mol Cell Cardiol, 33:1031–1040.

18.

Russell

and Proctor

. (2006). Small animal models of cardiovascular disease: tools for the study of the roles of metabolic syndrome, dyslipidemia, and atherosclerosis. Cardiovasc Pathol, 15:318–330.

19.

Elmadhun

, Sabe

, Robich

, Chu

, Lassaletta

and Sellke

. (2013). The pig as a valuable model for testing the effect of resveratrol to prevent cardiovascular disease. Ann N Y Acad Sci, 1290:130–135.

20.

Suzuki

, Yeung

and Ikeno

. (2011). The representative porcine model for human cardiovascular disease. J Biomed Biotechnol, 2011:195483.

21.

Koudstaal

, Jansen of Lorkeers

, Gho

, van Hout

, Jansen

, Grundeman

, Pasterkamp

, Doevendans

, Hoefer

and Chamuleau

. (2014). Myocardial infarction and functional outcome assessment in pigs. J Vis Exp [Epub ahead of print];, DOI: 10.3791/51269.

22.

Camacho

, Fan

, Liu

and He

. (2016). Large mammalian animal models of heart disease. J Cardiovasc Dev Dis, 3:30.

23.

, Chang

, Xiong

, Zhang

, Somasundaram

, Lepley

, Swingen

, Su

, et al. (2014). Cardiac repair in a porcine model of acute myocardial infarction with human induced pluripotent stem cell-derived cardiovascular cells. Cell Stem Cell, 15:750–761.

24.

, Minami

, Shiozaki

, Yu

, Yajima

, Miyagawa

, Shiba

, Morone

, Fukushima

, et al. (2017). Human pluripotent stem cell-derived cardiac tissue-like constructs for repairing the infarcted myocardium. Stem Cell Rep, 9:1546–1559.

25.

Mezrich

, Haller

, Arn

, Houser

, Madsen

and Sachs

. (2003). Histocompatible miniature swine: an inbred large-animal model. Transplantation, 75:904–907.

26.

Kwon

, Jeon

, Oh

, Ock

, Im

, Lee

, Im

, Lee

, Oh

, Park

and Hwang

. (2013). Generation of leukemia inhibitory factor-dependent induced pluripotent stem cells from the Massachusetts General Hospital miniature pig. Biomed Res Int, 2013:140639.

27.

Luo

, Qin

, Kural

, Schwan

, Li

, Bartulos

, Cong

, Ren

, Gui

, et al. (2017). Vascular smooth muscle cells derived from inbred swine induced pluripotent stem cells for vascular tissue engineering. Biomaterials, 147:116–132.

28.

Ezashi

, Telugu

, Alexenko

, Sachdev

, Sinha

and Roberts

. (2009). Derivation of induced pluripotent stem cells from pig somatic cells. Proc Natl Acad Sci U S A, 106:10993–10998.

29.

Esteban

, Xu

, Yang

, Peng

, Qin

, Li

, Jiang

, Chen

, Deng

, et al. (2009). Generation of induced pluripotent stem cell lines from Tibetan miniature pig. J Biol Chem, 284:17634–17640.

30.

Park

, Kim

, Uh

, Choi

, Kim

, Lee

, Yang

, Kim

, Ka

, Kim

and Lee

. (2013). Primed pluripotent cell lines derived from various embryonic origins and somatic cells in pig. PLoS One, 8:e52481.

31.

, Chen

, Ren

, Bao

, Liao

, Cui

, Rao

, Li

, Gu

, et al. (2009). Generation of pig induced pluripotent stem cells with a drug-inducible system. J Mol Cell Biol, 1:46–54.

32.

Kim

, Kwon

, Koo

, Moon

, Park

, Yum

, Lee

and Jang

. (2019). Production of transgenic porcine embryos reconstructed with induced pluripotent stem-like cells derived from porcine endogenous factors using piggyBac system. Cell Reprogram, 21:26–36.

33.

Congras

, Barasc

, Canale-Tabet

, Plisson-Petit

, Delcros

, Feraud

, Oudrhiri

, Hadadi

, Griscelli

, et al. (2016). Non integrative strategy decreases chromosome instability and improves endogenous pluripotency genes reactivation in porcine induced pluripotent-like stem cells. Sci Rep, 6:27059.

34.

Fukuda

, Tani

, Haraguchi

, Donai

, Nakajima

, Uenishi

, Eitsuka

, Miyagawa

, Song

, et al. (2017). Expression of six proteins causes reprogramming of porcine fibroblasts into induced pluripotent stem cells with both active X chromosomes. J Cell Biochem, 118:537–553.

35.

Strnadel

, Carromeu

, Bardy

, Navarro

, Platoshyn

, Glud

, Marsala

, Kafka

, Miyanohara

, et al. (2018). Survival of syngeneic and allogeneic iPSC-derived neural precursors after spinal grafting in minipigs. Sci Transl Med, 10:pii:eaam6651.

36.

, Nguyen

, Lee

, Xu

, Hu

, Plews

, Han

, Huber

, Lee

, et al. (2012). Microfluidic single-cell analysis shows that porcine induced pluripotent stem cell-derived endothelial cells improve myocardial function by paracrine activation. Circ Res, 111:882–893.

37.

Montserrat

, de Onate

, Garreta

, Gonzalez

, Adamo

, Eguizabal

, Hafner

, Vassena

and Izpisua Belmonte

. (2012). Generation of feeder-free pig induced pluripotent stem cells without Pou5f1. Cell Transplant, 21:815–825.

38.

Yuan

, Lee

, Park

, Spate

, Prather

, Wells

and Roberts

. (2014). Cell cycle synchronization of leukemia inhibitory factor (LIF)-dependent porcine-induced pluripotent stem cells and the generation of cloned embryos. Cell Cycle, 13:1265–1276.

39.

Nichols

and Smith

. (2009). Naive and primed pluripotent states. Cell Stem Cell, 4:487–492.

40.

Zhang

, Wei

, Zhang

, Li

, Liu

, Pu

, Li

, Cao

, et al. (2014). Efficient reprogramming of naive-like induced pluripotent stem cells from porcine adipose-derived stem cells with a feeder-independent and serum-free system. PLoS One, 9:e85089.

41.

Petkov

, Hyttel

and Niemann

. (2014). The small molecule inhibitors PD0325091 and CHIR99021 reduce expression of pluripotency-related genes in putative porcine induced pluripotent stem cells. Cell Reprogram, 16:235–240.

42.

, Yu

, Cai

and Wang

. (2018). Preserving self-renewal of porcine pluripotent stem cells in serum-free 3i culture condition and independent of LIF and b-FGF cytokines. Cell Death Discov, 4:21.

43.

Aravalli

, Cressman

and Steer

. (2012). Hepatic differentiation of porcine induced pluripotent stem cells in vitro. Vet J, 194:369–374.

44.

, Mich-Basso

, Lin

and Yang

. (2014). High efficient differentiation of functional hepatocytes from porcine induced pluripotent stem cells. PLoS One, 9:e100417.

45.

, Shan

, Wu

, Shen

, Liu

, Shen

, Liu

and Lei

. (2014). Generation of neural progenitors from induced Bama miniature pig pluripotent cells. Reproduction, 147:65–72.

46.

Gallegos-Cardenas

, Webb

, Jordan

, West

, Yang

, Wang

and Stice

. (2015). Pig induced pluripotent stem cell-derived neural rosettes developmentally mimic human pluripotent stem cell neural differentiation. Stem Cells Dev, 24:1901–1911.

47.

Webb

, Gallegos-Cardenas

, Miller

, Solomotis

, Liu

, West

and Stice

. (2017). Pig induced pluripotent stem cell-derived neural rosettes parallel human differentiation into sensory neural subtypes. Cell Reprogram, 19:88–94.

48.

Kim

, Kim

, Hwang

, Kim

, Lee

, Park

and Hyun

. (2019). Neural induction of porcine-induced pluripotent stem cells and further differentiation using glioblastoma-cultured medium. J Cell Mol Med [Epub ahead of print];, DOI: 10.1111/jcmm.14111.

49.

Genovese

, Domeier

, Telugu

and Roberts

. (2017). Enhanced development of skeletal myotubes from porcine induced pluripotent stem cells. Sci Rep, 7:41833.

50.

Zhou

, Wang

, Liu

, Fernandez de Castro

, Ezashi

, Telugu

, Roberts

, Kaplan

and Dean

. (2011). Differentiation of induced pluripotent stem cells of swine into rod photoreceptors and their integration into the retina. Stem Cells, 29:972–980.

51.

Zhao

, Zhang

, Rong

and Xu

. (2011). Immunogenicity of induced pluripotent stem cells. Nature, 474:212–215.

52.

West

, Uhl

, Liu

, Stowe

, Lu

, Yu

, Gallegos-Cardenas

, Pratt

and Stice

. (2011). Brief report: chimeric pigs produced from induced pluripotent stem cells demonstrate germline transmission and no evidence of tumor formation in young pigs. Stem Cells, 29:1640–1643.

53.

Choi

, Park

, Son

, Hwang

, Lee

, Ka

, Park

and Lee

. (2016). Reactivation of endogenous genes and epigenetic remodeling are barriers for generating transgene-free induced pluripotent stem cells in pig. PLoS One, 11:e0158046.

54.

Cheng

, Guo

, Li

, Liu

, Gao

, Cheng

, Hu

and Wang

. (2012). Porcine induced pluripotent stem cells require LIF and maintain their developmental potential in early stage of embryos. PLoS One, 7:e51778.

55.

Telugu

, Ezashi

, Sinha

, Alexenko

, Spate

, Prather

and Roberts

. (2011). Leukemia inhibitory factor (LIF)-dependent, pluripotent stem cells established from inner cell mass of porcine embryos. J Biol Chem, 286:28948–28953.

56.

, Feng

, Yu

, Wu

, Zou

, Ma

, Feng

, Huang

, Ouyang

, et al. (2015). Barriers for deriving transgene-free pig iPS cells with episomal vectors. Stem Cells, 33:3228–3238.

57.

West

, Terlouw

, Kwon

, Mumaw

, Dhara

, Hasneen

, Dobrinsky

and Stice

. (2010). Porcine induced pluripotent stem cells produce chimeric offspring. Stem Cells Dev, 19:1211–1220.

58.

Xiao

, Xie

, Cao

, Yu

, Xing

, Chen

, Musselman

, Xie

, West

, et al. (2012). Comparative epigenomic annotation of regulatory DNA. Cell, 149:1381–1392.

59.

Xie

, Zhang

, Wei

, Li

, Weng

, Kong

and Liu

. (2016). Histone H3 lysine 27 trimethylation acts as an epigenetic barrier in porcine nuclear reprogramming. Reproduction, 151:9–16.

60.

Mao

, Zhang

, Deng

, Wang

, Liu

, Fu

, Zhao

, Wang

and Liu

. (2017). Epigenetic modifiers facilitate induction and pluripotency of porcine iPSCs. Stem Cell Rep, 8:11–20.

61.

Hou

, Li

, Zhang

, Liu

, Guan

, Li

, Zhao

, Ye

, Yang

, et al. (2013). Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science, 341:651–654.

62.

Zhao

, Shi

, Zhou

, He

, Yang

and Wu

. (2018). DZNep and UNC0642 enhance in vitro developmental competence of cloned pig embryos. Reproduction, 157:359–369.

63.

Liu

, Cai

, Wang

, Nie

, Zhang

, Xu

, Zhang

, Lu

, Wang

, Poo

and Sun

. (2018). Cloning of macaque monkeys by somatic cell nuclear transfer. Cell, 174:245.

64.

Gao

, Nowak-Imialek

, Chen

, Herrmann

, Ruan

, Chen

ACH

, Eckersley-Maslin

, Ahmad

, et al. (2019). Establishment of porcine and human expanded potential stem cells. Nat Cell Biol, 21:687–699.

65.

Lupu

and Storb

. (2007). Five decades of progress in haematopoietic cell transplantation based on the preclinical canine model. Vet Comp Oncol, 5:14–30.

66.

Power

and Tonkin

. (1999). Large animal models of heart failure. Aust N Z J Med, 29:395–402.

67.

Rowell

, McCarthy

and Alvarez

. (2011). Dog models of naturally occurring cancer. Trends Mol Med, 17:380–388.

68.

Silbergleit

. (2006). Norman E. Shumway and the early heart transplants. Tex Heart Inst J, 33:274–275; author reply 275.

69.

Dixon

and Spinale

. (2009). Large animal models of heart failure: a critical link in the translation of basic science to clinical practice. Circ Heart Fail, 2:262–271.

70.

Ostrander

, Galibert

and Patterson

. (2000). Canine genetics comes of age. Trends Genet, 16:117–124.

71.

Garibal

, Hollville

, Bell

, Kelly

, Renouf

, Kawaguchi

, Rickinson

and Wiels

. (2007). Truncated form of the Epstein-Barr virus protein EBNA-LP protects against caspase-dependent apoptosis by inhibiting protein phosphatase 2A. J Virol, 81:7598–7607.

72.

Webb

, Jeffery

, Olby

and Muir

. (2004). Behavioural analysis of the efficacy of treatments for injuries to the spinal cord in animals. Vet Rec, 155:225–230.

73.

Blight

, Toombs

, Bauer

and Widmer

. (1991). The effects of 4-aminopyridine on neurological deficits in chronic cases of traumatic spinal cord injury in dogs: a phase I clinical trial. J Neurotrauma, 8:103–119.

74.

Jeffery

, Smith

, Lakatos

, Ibanez

, Ito

and Franklin

. (2006). Clinical canine spinal cord injury provides an opportunity to examine the issues in translating laboratory techniques into practical therapy. Spinal Cord, 44:584–593.

75.

Levine

, Levine

, Porter

, Topp

and Noble-Haeusslein

. (2011). Naturally occurring disk herniation in dogs: an opportunity for pre-clinical spinal cord injury research. J Neurotrauma, 28:675–688.

76.

Shimada

, Nakada

, Hashimoto

, Shigeno

, Shionoya

and Nakamura

. (2010). Generation of canine induced pluripotent stem cells by retroviral transduction and chemical inhibitors. Mol Reprod Dev, 77:2.

77.

Lee

, Xu

, Plews

, Nguyen

, Nag

, Lyons

, Han

, Hu

, Lan

, et al. (2011). Preclinical derivation and imaging of autologously transplanted canine induced pluripotent stem cells. J Biol Chem, 286:32697–32704.

78.

Luo

, Suhr

, Chang

, Wang

, Ross

, Nelson

, Venta

, Knott

and Cibelli

. (2011). Generation of leukemia inhibitory factor and basic fibroblast growth factor-dependent induced pluripotent stem cells from canine adult somatic cells. Stem Cells Dev, 20:1669–1678.

79.

Luo

and Cibelli

. (2016). Conserved role of bFGF and a divergent role of LIF for pluripotency maintenance and survival in canine pluripotent stem cells. Stem Cells Dev, 25:1670–1680.

80.

Greber

, Wu

, Bernemann

, Joo

, Han

, Ko

, Tapia

, Sabour

, Sterneckert

, Tesar

and Scholer

. (2010). Conserved and divergent roles of FGF signaling in mouse epiblast stem cells and human embryonic stem cells. Cell Stem Cell, 6:215–226.

81.

Goncalves

NJN

, Bressan

, Roballo

KCS

, Meirelles

, Xavier

PLP

, Fukumasu

, Williams

, Breen

, Koh

, et al. (2017). Generation of LIF-independent induced pluripotent stem cells from canine fetal fibroblasts. Theriogenology, 92:75–82.

82.

Whitworth

, Ovchinnikov

and Wolvetang

. (2012). Generation and characterization of LIF-dependent canine induced pluripotent stem cells from adult dermal fibroblasts. Stem Cells Dev, 21:2288–2297.

83.

Nishimura

, Hatoya

, Kanegi

, Sugiura

, Wijewardana

, Kuwamura

, Tanaka

, Yamate

, Izawa

, et al. (2013). Generation of functional platelets from canine induced pluripotent stem cells. Stem Cells Dev, 22:2026–2035.

84.

Whitworth

, Frith

, Ovchinnikov

, Cooper-White

and Wolvetang

. (2014). Derivation of mesenchymal stromal cells from canine induced pluripotent stem cells by inhibition of the TGFbeta/activin signaling pathway. Stem Cells Dev, 23:3021–3033.

85.

Chow

, Johnson

, Regan

, Wheat

, Webb

, Koch

and Dow

. (2017). Safety and immune regulatory properties of canine induced pluripotent stem cell-derived mesenchymal stem cells. Stem Cell Res, 25:221–232.

86.

Nishimura

, Hatoya

, Kanegi

, Wijesekera

DPH

, Sanno

, Tanaka

, Sugiura

, Hiromitsu Tamada

, Imai

and Inaba

. (2017). Feeder-independent canine induced pluripotent stem cells maintained under serum-free conditions. Mol Reprod Dev, 84:329–339.

87.

Tsukamoto

, Nishimura

, Yodoe

, Kanegi

, Tsujimoto

, Alam

, Kuramochi

, Kuwamura

, Ohtaka

, et al. (2018). Generation of footprint-free canine induced pluripotent stem cells using auto-erasable sendai virus vector. Stem Cells Dev, 27:1577–1586.

88.

Allen

, Hankenson

, Goodrich

, Boivin

and von Rechenberg

. (2017). Ethical use of animal models in musculoskeletal research. J Orthop Res, 35:740–751.

89.

Dias

, Camassa

, Bordelo

, Babo

, Viegas

, Dourado

, Reis

and Gomes

. (2018). Preclinical and translational studies in small ruminants (sheep and goat) as models for osteoporosis research. Curr Osteoporos Rep, 16:182–197.

90.

Breton

, Sharma

, Diaz

, Parham

, Graham

, Neil

, Whitelaw

, Milne

and Donadeu

. (2013). Derivation and characterization of induced pluripotent stem cells from equine fibroblasts. Stem Cells Dev, 22:611–621.

91.

Nagy

, Sung

, Zhang

, Laflamme

, Vincent

, Agha-Mohammadi

, Woltjen

, Monetti

, Michael

, Smith

and Nagy

. (2011). Induced pluripotent stem cell lines derived from equine fibroblasts. Stem Cell Rev, 7:693–702.

92.

Sharma

, Livesey

, Wyllie

, Proudfoot

, Whitelaw

, Hay

and Donadeu

. (2014). Generation of functional neurons from feeder-free, keratinocyte-derived equine induced pluripotent stem cells. Stem Cells Dev, 23:1524–1534.

93.

Lee

, Kim

, Lee

, Park

, Cho

, Kim

and Jeong

. (2016). Generation of equine-induced pluripotent stem cells and analysis of their therapeutic potential for muscle injuries. Cell Transplant, 25:2003–2016.

94.

Amilon

, Cortes-Araya

, Moore

, Lee

, Lillico

, Breton

, Esteves

and Donadeu

. (2018). Generation of functional myocytes from equine induced pluripotent stem cells. Cell Reprogram, 20:275–281.

95.

Nagy

and Nagy

. (2015). Derivation of equine-induced pluripotent stem cell lines using a piggyBac transposon delivery system and temporal control of transgene expression. Methods Mol Biol, 1330:79–88.

96.

Whitworth

, Ovchinnikov

, Sun

, Fortuna

and Wolvetang

. (2014). Generation and characterization of leukemia inhibitory factor-dependent equine induced pluripotent stem cells from adult dermal fibroblasts. Stem Cells Dev, 23:1515–1523.

97.

Aguiar

, Therrien

, Lemire

, Segura

, Smith

and Theoret

. (2016). Differentiation of equine induced pluripotent stem cells into a keratinocyte lineage. Equine Vet J, 48:338–345.

98.

Quattrocelli

, Giacomazzi

, Broeckx

, Ceelen

, Bolca

, Spaas

and Sampaolesi

. (2016). Equine-induced pluripotent stem cells retain lineage commitment toward myogenic and chondrogenic fates. Stem Cell Rep, 6:55–63.

99.

Baird

, Dominguez-Falcon

, Saeed

and Guest

. (2019). Biocompatible 3D printed thermoplastic scaffolds for osteoblast differentiation of equine iPS cells. Tissue Eng Part C Methods, 25:253–261.

100.

Lepage

, Nagy

, Sung

, Kandel

, Nagy

and Koch

. (2016). Generation, characterization, and multilineage potency of mesenchymal-like progenitors derived from equine induced pluripotent stem cells. Stem Cells Dev, 25:80–89.

101.

Fortuna

PRJ

, Bielefeldt-Ohmann

, Ovchinnikov

, Wolvetang

and Whitworth

. (2018). Cortical neurons derived from equine induced pluripotent stem cells are susceptible to neurotropic flavivirus infection and replication: an in vitro model for equine neuropathic diseases. Stem Cells Dev, 27:704–715.

102.

Liu

, Balehosur

, Murray

, Kelly

, Sumer

and Verma

. (2012). Generation and characterization of reprogrammed sheep induced pluripotent stem cells. Theriogenology, 77:338–346.e1.

103.

Tai

, Liu

, Gao

, Jin

, Xu

, Zuo

, Liang

and Liu

. (2015). Generation of Arbas Cashmere goat induced pluripotent stem cells through fibroblast reprogramming. Cell Reprogram, 17:297–305.

104.

German

, Campbell

, Thornton

, McLachlan

, Sweetman

and Alberio

. (2015). Ovine induced pluripotent stem cells are resistant to reprogramming after nuclear transfer. Cell Reprogram, 17:19–27.

105.

Choi

, Lee

, Kim

, Woo

, Kim

and Lee

. (2019). Chemically defined media can maintain pig pluripotency network in vitro. Stem Cell Rep, 13:221–234.

106.

Chowdhury

, Li

, Poh

, Yokohama-Tamaki

, Wang

and Tanaka

. (2010). Soft substrates promote homogeneous self-renewal of embryonic stem cells via downregulating cell-matrix tractions. PLoS One, 5:e15655.

107.

Chowdhury

, Na

, Li

, Poh

, Tanaka

, Wang

and Wang

. (2010). Material properties of the cell dictate stress-induced spreading and differentiation in embryonic stem cells. Nat Mater, 9:82–88.

108.

Deuse

, Hu

, Gravina

, Wang

, Tediashvili

, De

, Thayer

, Wahl

, Garcia

, et al. (2019). Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients. Nat Biotechnol, 37:252–258.

109.

Gornalusse

, Hirata

, Funk

, Riolobos

, Lopes

, Manske

, Prunkard

, Colunga

, Hanafi

, et al. (2017). HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells. Nat Biotechnol, 35:765–772.