Review Article Authors
Over the past 25 years, the reverse genetic approach including precise and conditional replacement or loss of gene function at a specific locus was considered possible only in mice due to the absence of embryonic stem (ES) or induced pluripotent stem (iPS) cell lines in other species. Recently, however, stem cell technology in rats has become available for biomedical research. In this paper we overview the recent progress of rat ES and iPS cell technology. Starting from the establishment of rat ES cells, the use of ES cells for foreign gene transfer and endogenous gene knock-out is discussed, followed by the successful establishment of rat iPS cells and the generation of an iPS cell-derived organ via interspecific blastocyst complementation. Finally, the possible contribution of rat stem cell technology to reproductive medicine is described.
Rats (Rattus norvegicus) have been used more extensively than mice in the research fields of neuroscience, pharmacology and toxicology. There are more than 100 rat strains with various genetic backgrounds, including some useful models for human diseases (e.g., SHR and BB for hypertension and diabetes, respectively), as well as the uncountable number of transgenic rat strains. Rats have the advantage of being a reasonably well-characterized and intermediate-sized rodent that can be maintained more cheaply than larger animals and can often be manipulated more easily than smaller rodents.
Although several technologies have been applied to modify rat genomes , the reverse genetic approach (precise and conditional replacements [knock-in] or loss of gene function [knock-out] at a specific locus) was considered impossible in rats because any protocols to establish stem cell lines conventionally used in mice [4, 5] were not applicable to rats. Recently, however, functional germline-competent embryonic stem (ES) cell lines [6, 7] and induced pluripotent stem (iPS) cell lines [8, 9] have been reported for this species. General advantages and disadvantages of ES and iPS cells are summarized in Table 1. In this paper we discuss the recent progress of rat ES/iPS cell technology.
The breakthrough in establishing rat ES cell lines was at the end of 2008. Functional germline-competent ES cell lines were reported by using a few inhibitors for fibroblast growth factor (FGF) receptor, mitogen activated protein kinase kinase (MEK) and glycogen synthase kinase 3 (GSK3) in differentiation-related signaling pathways [6, 7]. This protocol, the so-called 3i/2i culture system, originated from ES cell research in mice  and proved reproducible even after slight modifications were added to the culture system [11, 12]. The modification made by Hirabayashi et al.  is to replace MEK activation inhibitor PD1843521 with MEK inhibitor PD325901 and to add rat leukemia inhibitory factor (LIF) (ESGRO) instead of LIF-secreting feeder cells to the 3i culture system, while that made by Kawamata and Ochiya  is to add fetal bovine serum (FBS), -mercaptoethanol, rat LIF, and inhibitors for Rho-associated coiled-coil kinase and transforming growth factor- type-I receptor ALK5 kinase (Y-27632 and A-83-01, respectively) to the 2i culture medium containing MEK and GSK3 inhibitors (PD325901 and CHIR99021, respectively).
The rat ES cell lines established by Hirabayashi et al.  are described here in detail. Blastocysts at E4.5 were recovered from Wistar females copulated with a homogenous CAG/venus transgenic male rat (green fluorescence of the venus gene was used as the transgenic marker). Zona-free blastocysts were placed on mitomycin C (MMC)-treated mouse embryonic fibroblasts (MEF). The culture medium consisted of 2 M FGF receptor inhibitor (SU5402), 1 M MEK inhibitor (PD0325901), 3 M GSK3 inhibitor (CHIR99021) and 1,000 U/ml rat LIF (ESGRO) in N2B27 medium. After 7 days of culture, the outgrowths of the blastocysts (Fig. 1a) were disaggregated by gentle pipetting and transferred to the same MEF/3i conditions (first passage). When ES cell-like colonies emerged (Fig. 1b), they were trypsinized and then expanded (Fig. 1c). The tentative ES cell lines were maintained in MEF/3i conditions, with medium exchange every other day and trypsinization/expansion (passage) every 3 days. Otherwise, the ES cell lines were cryopreserved to prevent senescence.
Establishment of rat ES cell line . a Outgrowth of a blastocyst on MEF feeders 7 days after plating. b Formation of colonies 3 days after the first passage. c Expanded ES cell colonies 2 days after fourth passage. d Alkaline phosphatase-positive colony at passage 12. e ES cells at passage 17. Under the daily exchange of culture medium, very few cells showed any signs of differentiation. f ES cells at passage 17, maintained with medium exchange every other day. Differentiated extraembryonic cells were observed. Scale barsa, e, f 100 m, b c, d 500 m
It was checked whether the delivered cells were alkaline phosphatase (AP)-positive (Fig. 1d). Nine ES cell lines (69.2%) were established from 13 transgenic blastocysts. Among them, two lines with excellent growth rate (rESWIv3i-1 and rESWIv3i-5) were selected, and both lines were found to be female by PCR analysis to detect rat Sry gene. Attachment of ES cell colonies with the feeder cells was not as strong, and the morphological appearance of the rat ES cell colonies was similar to that of mouse ES cells. As the passage number of the ES cells increased, signs of differentiation into extraembryonic cell-like cells were observed in cultures, especially when the culture medium was exchanged every other day rather than every day (Fig. 1e, f). In addition, expression of stem cell marker genes, such as Oct4, Nanog, Fgf4 and Rex1, was confirmed by reverse transcriptionpolymerase chain reaction (RT-PCR) analysis in rESWIv3i-1 and rESWIv3i-5 lines. Furthermore, multipotency of the tentative ES cells was investigated by subcutaneous transplantation of the rESWIv3i-1 cells into an adult male F344 nude rat. Five weeks after the transplantation, a tumor was observed. By histological analysis, the tumor was found to be a teratoma with various tissues including gut-like epithelium or hepatic cells (endoderm), bone, cartilage or muscle (mesoderm), and neural tissues (ectoderm).
To generate ES cell-derived chimeras, host blastocysts at E4.5 derived from Wistar females or Wistar Dark Agouti (DA) F1 females were each microinjected with 10 ES cells at passages 68 (Fig. 2a, b). Collapsed blastocysts (Fig. 2c) were re-blasturated 12 h after the microinjection, and allowed to develop to fetus (E15.5) or full-term pups in pseudopregnant Wistar recipients. All of the E15.5 fetuses (100%; Fig. 2d) and the majority of the newborn pups (81.8100%) were chimeric and expressed the venus gene. The characteristics of the ES cells was successfully transmitted to their next generation in both lines. The ability of the ES cells to participate in chimeras was still high (78.6100%) at advanced passage numbers (17 or 18). Overall efficiency of producing chimeric rats (50.3%, 94 chimeras/187 injected embryos) was higher than 8.2% (20/245) as reported in Buehr et al.  and 11.0% (26/237) in Li et al. . This higher efficiency of chimera rat production is probably due to the rat strain combination used for donor (ES cells) and host (blastocysts) and/or modification of culture medium.
Production of chimeric rats with ES cells . a Microinjection of 10 ES cells into a blastocoele of E4.5 blastocyst. b Semi-bright, fluorescent image of venus-positive ES cells in the blastocyst. c Collapsed blastocysts immediately after microinjection. d Venus-positive fetal rat at E15.5
Functional germline-competent rat ES cell lines were established by applying the 2i/3i culture system, and the minimal essential materials for conducting transgenic studies including reverse genetic approaches, were now ready to use in rats. We now describe the production efficiency of chimeric rats by blastocyst injection of ES cells electroporated with a humanized Kusabira-Orange (huKO) gene and the germline transmission of the huKO gene from the chimeras to the next generation .
Rat ES cell lines were established from E4.5 blastocysts derived from BrownNorway (BN) females copulated with BN males, as described above, by applying the 2i (SU5402-free) culture system. One of the established lines, named as rESBN2i-4, was derived from a male embryo, based on PCR analysis. At passage 8, the ES cells in the N2B27 medium supplemented with 10% FBS (1 106 cells/0.5 ml) were electroporated with 25 g huKO gene (CAG/huKO-neo plasmid; 7.5 kb, Fig. 3a). The electroporated ES cells were placed in 3 ml of 2i medium + 5% FBS (passage 9), and the next day the medium was changed to serum-free 2i medium. Two days after the electroporation, G418 (200 g/ml) was added to the medium. The number of neomycin-resistant colonies (passage 10) was counted 8 days after electroporation. The huKO-positive ES cells were passaged twice in G418-free medium. Host blastocysts derived from Wistar/ST or WistarHannover females were each microinjected with 10 of the G418-resistant huKO-positive ES cells, and allowed to develop to full-term in pseudopregnant Wistar recipients. Transfer of 116 Wistar/ST blastocysts and 97 WistarHannover blastocysts resulted in 31 and 44 new-born offspring (26.7% and 45.4%), and 22 (70.9%; male 12, female 10) and 34 (77.3%; male 15, female 17, not-identified 2) out of the offspring were judged as chimeras by their coat color, respectively (Fig. 3b). Using non-electroporated control ES cells, a similar offspring rate (37.5%, 9/24) and chimera production efficiency (88.9%, 8/9) were obtained. Rat strain for host blastocysts may be a factor influencing the overall efficiency of chimera production, due to different preference for full-term development. Germline transmission of the CAG/huKO-neo gene was confirmed in 6 out of 25 G1 offspring (Fig. 3c) derived from 1 chimeric male with > 95% brown-colored coat. Thus, integration of exogenous DNA into rat ES cells did not affect the production efficiency of chimera offspring. The result described in this section  achieved the first successful production of transgenic rats via electroporated ES cells, followed by the work of Kawamata and Ochiya .
Rat transgenesis with ES cells . a The construction of CAG/huKO-neo plasmid; 7.5 kb. b Chimeric rats with different contribution of brown-colored coat at G0 generation, 17 days old. c A huKO transgenic offspring (brown-colored) with huKO-negative littermates, 3 weeks old
Although knock-out rats were also successfully produced by N-ethyl-N-nitrosourea-induced transgenesis , sleeping beauty transposon-tagged mutagenesis , or zinc-finger nuclease-based transgenesis [3, 14], such successes may not have an impact on creating rat models for human diseases due to limitations in genome modification by these technologies. In this section, we describe the successful production of endogenous p53 gene knock-out rats by homologous recombination in ES cells .
p53, consisting of ten exons with the translation start codon located within exon 2, is a tumor suppressor gene located on rat chromosome 10, and mutations in the p53 gene are highly associated with genetic lesions in human cancers. Two male lines of ES cells were established from DA blastocysts by the 2i culture system and named as DAc4 and DAc8, respectively. After electroporation with CAG-EGFP-IRES-Pac vector (6.7 kb 5 and 1.6 kb 3 homology arms) to replace exons 25 of p53, successful targeting of the p53 gene occurred at 1.1% (4/356) and 3.7% (10/270) in the DAc4 line at passage 32 and the DAc8 line at passage 14, respectively. One correctly targeted ES cell colony, named as DAc8-p53-1, was microinjected into E4.5 F344 blastocysts (n = 79) and resulted in the birth of 24 live pups (30.4%). Among these 24 pups, 16 (75.0%; male 10, female 6) were chimeras with agouti coat color and all the male chimeras were fertile. However, examination of their >600 G1 offspring failed to show production of p53 targeted rats. The authors  suggest that this failure was caused by chromosomal abnormalities in the ES cells, because over 65% of the DAc8-p53-1 rat ES cells were found to be polyploid by karyotype analysis. Subcloning of the DAc8-p53-1 rat ES cells resulted in the appearance of round and compact colonies (approximately 10%). Among 20 subclones harvested, two (10.0%) were identified to carry euploid chromosome numbers (2n = 42). After microinjection of the subcloned ES cells into 39 F344 blastocysts and transfer to pseudopregnant SpragueDawley (SD) recipients, two male chimeras were produced and one of the two was germline chimera. Germline transmission of the GFP gene was confirmed in 6 out of the 76 G1 offspring. Three of the germline pups (male 1, female 2) were identified by genotyping and Southern blot analysis to be p53 heterozygotic rats carrying one wild-type allele and one targeted p53 allele. Intercrossing of the three p53 heterozygous rats resulted in the production of 12 pups, of which 9 (75.0%) were GFP-positive. Genotyping showed that 2 of the 9 pups were p53 homozygous (knock-out) rats, with further confirmation by Northern and Western blot analyses (absence of p53 mRNA and protein, respectively). Thus, gene targeting via homologous recombination in rat ES cells and the production of knock-out rats were achieved for the first time.
iPS cells are a type of pluripotent stem cell, similar to ES cells, that are artificially delivered from a non-pluripotent somatic cell by inducing a forced expression of specific genes. iPS cells can be established without the controversial use of blastocyst-stage embryos. iPS cells also have the advantage of avoiding the issue of graft-versus-host disease and immune rejection, if they are delivered entirely from the patient. The genes to be transfected into a somatic cell with a viral vector, the so-called Yamanaka factors, include Oct4, Sox2, c-Myc, and Klf4 [16, 17]. The Oct4 and Sox2 have been identified as crucial transcriptional regulators, both of which play a key role in maintaining pluripotency. The Klf4 is also the transcriptional regulator gene and is involved in cell proliferation, differentiation and survival. The c-Myc is the oncogene encoding for protein that binds to the DNA of other genes, and thus acts as a transcription factor. The induction efficiency of iPS cells (reprogramming efficiency of differentiated somatic cells) was 10-times higher when embryonic fibroblasts rather than tail tissue-derived fibroblasts (adult somatic cells) were used for transfection, and the morphology of iPS cell colonies was similar to that of ES cell colonies . Once the iPS cells have been established, the retroviral or lentiviral transgenes become silenced theoretically, and the endogenous genes encoding these factors become activated. However, for the future therapeutic application of iPS cells, the viral transfection system and the use of oncogene c-Myc with the risk of tumor formation need to be replaced by alternative induction methods. The disadvantages of the original protocol have already been overcome by the use of adenovirus  or plasmid  as the vector, the direct introduction of proteins encoded by Yamanaka factors , and the elimination of the c-Myc gene from the cocktails , but the induction efficiency of iPS cells by such alternative methods may be unsatisfactory and should be improved further.
The first successful establishment of rat iPS cell lines was published from two independent laboratories in 2009 [8, 9]. Both protocols employed to establish the iPS cells were theoretically the same as, but slightly modified from, that reported by Takahashi and Yamanaka  where Yamanaka factors (Oct4, Sox2, c-Myc, and Klf4) were designed in a retroviral vector for transfection into adult somatic cells. The commercially available construction of the pMXs retroviral vector typical for the transfection of the factors  is shown in Fig. 4. The protocol by Li et al.  includes the construction of a retroviral vector with Oct4, Sox2 and Klf4 (c-Myc-free Yamanaka factors) and the transfection into liver progenitor cells prepared from WB-F344 rats. On the other hand, Liao et al.  used the lentiviral vector constructed with all four Yamanaka factors for transfection into primary ear fibroblasts prepared from 10-week-old SD rats, because transfection with retroviral vectors resulted in the failure of harvesting the iPS cell-like colonies. Both groups identified rat iPS cell-like colonies from the cultures in ES medium (Knockout; DMEM medium supplemented with 1020% KnockOut serum replacer, 0.1 mM non-essential amino acids, 1 mM l-glutamine, and 0.1 mM -mercaptoethanol) until 10 days after the viral transfection. The expression of stem cell marker genes (such as rat Oct4, Sox2, Nanog) in the iPS cells and the contribution to teratoma formation from the iPS cells were confirmed. Liao et al.  did not describe the production of chimeric rats using their iPS cells that were maintained on MEF in FBS-supplemented ES medium. In contrast, Li et al.  reported the successful production of chimeric rats by blastocyst injection of their iPS cells that were maintained in the presence of 0.5 M PD325901 (MEK inhibitor), 0.5 M A-83-01 (ALK5 inhibitor) and 3 M CHIR99021 (GSK3 inhibitor). The overall efficiency of producing chimeric rats was 16.7% (3 chimeras/18 injected embryos). The modified 3i culture system was known to support the self-renewal of mouse ES cells in a more robust manner [23, 24], but even after short-term culture a considerable amount of rat iPS cells exhibited spontaneous differentiation . Transmission of the iPS cell genetic characteristics through the germline was not investigated in their studies [8, 9].
Retroviral vectors (pMXs) designed to transfect four transcriptional genes, the so-called Yamanaka factors (Oct4, Sox2, c-Myc, and Klf4), into somatic cells . SD splicing donor, SA splicing acceptor, LTR long terminal repeats, MMLV Moloney murine leukemia virus
Therapeutic application of iPS cells includes the transplantation of differentiated cells, tissues or organs that can be regenerated from the stem cells. However, the in vitro production of tissue or organ with a three-dimensional structure from iPS cells is still difficult to achieve, despite the recent progress on the interactions of stem cells with growth factors and scaffolds. The idea for organ regeneration under an in vivo condition by blastocyst complementation was originally derived from Chen et al. . Mouse blastocysts from a Rag2/ mutant strain lacking matured lymphocytes were microinjected with wild-type mouse ES cells and the resultant Rag2//ES chimera offspring produced fully matured T- and B-lymphocytes that were originated from the ES cells (Fig. 5). This strategy of intraspecies blastocyst complementation was expanded to interspecies approach. If the rescue of organ deficiency in mouse by rat iPS cells was proven effective, the interspecies blastocyst complementation may be applicable for a combination of pig (host animal) and human (iPS cell origin).
Intraspecies and interspecies blastocyst complementation for organ regeneration from pluripotent stem cells. Chen et al.  reported an intraspecies assay that enabled mouse ES cells to differentiate into mature lymphocytes in Rag2/ mice (mature lymphocyte-deficient). Kobayashi et al.  reported an interspecies assay in which rat iPS cells formed a fully functional rat pancreas when injected into mouse blastocysts lacking the Pdx1 gene required for pancreas formation
The rat iPS cell line (named as riPS#3) used for the interspecies blastocyst complementation  was established from primary embryonic fibroblasts prepared from E14.5 Crlj:Wistar fetuses. Ten to 15 iPS cells were microinjected into each of the blastocysts derived from Pdx1+/ Pdx1/ mice. The Pdx1 gene is responsible for the formation of functional pancreas, and the pancreas in the Pdx1/ male founder arose principally from exogenous mouse iPS cells. Therefore, half of the mice born from the blastocysts were expected to be Pdx1/. Transfer of 139 blastocysts into recipient mice resulted in the birth of 34 offspring (24.5%), and among these offspring, 5 and 10 neonates were found to be Pdx1+/ and Pdx1/ chimeric mice with rat iPS cell-derived pancreatic epithelial cells, respectively, by the enhanced green fluorescence protein (EGFP). Function of the pancreas derived from the rat iPS cells was confirmed by immunohistological analyses for the presence of EGFP, -amylase, insulin, glucagon, and somatostatin. Development into adulthood (8 weeks old) of the wild-type Pdx1/ mice was uncommon, but two Pdx1/ chimeras complemented with the rat iPS cells survived to the adulthood stage. They secreted insulin in response to glucose loading, and maintained a normal serum glucose level. Thus, Kobayashi et al.  demonstrated that rat iPS cells can rescue organ deficiency in mice, as the rat iPS cells formed a fully functional pancreas when injected into mouse blastocysts lacking the Pdx1 gene required for pancreas formation.
A few approaches are promising to produce offspring exclusively derived from stem cells, in addition to the conventional, time-consuming method via chimera (Table 2). Tetraploid embryos are able to implant in the endometrium and form extraembryonic tissues such as placenta, without participating into fetal development. In mice, tetraploid blastocysts complemented with ES/iPS cells can develop into full-term offspring with a phenotype of the ES/iPS characteristics [27, 28]. This approach, once found reproducible and applicable to other species, would allow the generation of different types of organs from the stem cells at the same time (showing ultimate pluripotency of the stem cells). The attempt at tetraploid blastocyst complementation with rat ES/iPS cells is, however, a challenging endeavor at the present stage . Transplantation of stem cell nuclei into enucleated oocytes (cloning) is an alternative approach to produce animals derived from stem cells alone. Wakayama et al.  reported that 29% of mouse oocytes microinjected with ES cell nuclei developed into morulae/blastocysts and 8% of these embryos developed to full-term. Successful production of cloned rats with somatic cells was first reported by Zhou et al. . However, the production of rat offspring exclusively derived from ES/iPS cells via nuclear transplantation has not been very practical, because the reproducibility of the data on rat cloning remained questionable .
Stem cell technology in rats can also contribute to the field of reproductive medicine, because germ cells derived from the stem cells were detected in xenogenic chimeras (mouserat chimeras). Isotani et al.  recently reported that rat ES cells injected into nu/nu mouse blastocysts could contribute to form not only thymus but also sperm-like germ cells. Although the minimal essential techniques for microinsemination, such as intracytoplasmic sperm injection and round spermatid injection, are almost available in the rat , the functional normality of the rat germ cell-like cells observed in such xenogenic chimeras has not yet been confirmed. Nevertheless, the interspecies blastocyst complementation system using mutant mice or experimentally nitch-induced mice and pluripotent stem cells of the rats would provide an appropriate model for generation of human germ cells in the body of non-human species.
Functional germline-competent rat ES/iPS cell lines have been established and successfully applied to the production of gene-modified rats as well as whole organ regeneration with a three-dimensional structure. Culturing of blastocysts with a few inhibitors for FGF receptor, MEK and GSK3 in differentiation-related signaling pathways (2i/3i system) was the key essential for ES cell establishment, and a forced expression of transcription-regulating genes as Yamanaka factors (Oct4, Sox2, Klf4 and/or c-Myc) in somatic cells played an important role in iPS cell establishment. The widespread use of the rat ES/iPS cells would provide a practical breakthrough for a variety of biomedical research in the rats. The accumulation of basic and practical knowledge in this system may be useful in improving the ultimate therapeutic performance against the most severe forms of male infertility in humans.
The Japan Society for Reproductive Medicine
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