Ikuo Tomioka

Generation of transgenic non-human primates with germline transmission

The common marmoset (Callithrix jacchus) is increasingly attractive for use as a non-human primate animal model in biomedical research. It has a relatively high reproduction rate for a primate, making it potentially suitable for transgenic modification. Although several attempts have been made to produce non-human transgenic primates, transgene expression in the somatic tissues of live infants has not been demonstrated by objective analyses such as polymerase chain reaction with reverse transcription or western blots. Here we show that the injection of a self-inactivating lentiviral vector in sucrose solution into marmoset embryos results in transgenic common marmosets that expressed the transgene in several organs. Notably, we achieved germline transmission of the transgene, and the transgenic offspring developed normally. The successful creation of transgenic marmosets provides a new animal model for human disease that has the great advantage of a close genetic relationship with humans. This model will be valuable to many fields of biomedical research.

Generating induced pluripotent stem cells from common marmoset (Callithrix jacchus) fetal liver cells using defined factors, including Lin28.

Although embryonic stem (ES) cell–like induced pluripotent stem (iPS) cells have potential therapeutic applications in humans, they are also useful for creating genetically modified human disease models in nonhuman primates. In this study, we generated common marmoset iPS cells from fetal liver cells via the retrovirus‐mediated introduction of six human transcription factors: Oct‐3/4, Sox2, Klf4, c‐Myc, Nanog, and Lin28. Four to five weeks after introduction, several colonies resembling marmoset ES cells were observed and picked for further expansion in ES cell medium. Eight cell lines were established, and validation analyses of the marmoset iPS cells followed. We detected the expression of ES cell–specific surface markers. Reverse transcription‐PCR showed that these iPS cells expressed endogenous Oct‐3/4, Sox2, Klf4, c‐Myc, Nanog and Lin28 genes, whereas all of the transgenes were silenced. Karyotype analysis showed that two of three iPS cell lines retained a normal karyotype after a 2‐month culture. Both embryoid body and teratoma formation showed that marmoset iPS cells had the developmental potential to give rise to differentiated derivatives of all three primary germ layers. In summary, we generated marmoset iPS cells via the transduction of six transcription factors; this provides a powerful preclinical model for studies in regenerative medicine.

Efficient derivation of multipotent neural stem/progenitor cells from non-human primate embryonic stem cells.

The common marmoset (Callithrix jacchus) is a small New World primate that has been used as a non-human primate model for various biomedical studies. We previously demonstrated that transplantation of neural stem/progenitor cells (NS/PCs) derived from mouse and human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) promote functional locomotor recovery of mouse spinal cord injury models. However, for the clinical application of such a therapeutic approach, we need to evaluate the efficacy and safety of pluripotent stem cell-derived NS/PCs not only by xenotransplantation, but also allotransplantation using non-human primate models to assess immunological rejection and tumorigenicity. In the present study, we established a culture method to efficiently derive NS/PCs as neurospheres from common marmoset ESCs. Marmoset ESC-derived neurospheres could be passaged repeatedly and showed sequential generation of neurons and astrocytes, similar to that of mouse ESC-derived NS/PCs, and gave rise to functional neurons as indicated by calcium imaging. Although marmoset ESC-derived NS/PCs could not differentiate into oligodendrocytes under default culture conditions, these cells could abundantly generate oligodendrocytes by incorporating additional signals that recapitulate in vivo neural development. Moreover, principal component analysis of microarray data demonstrated that marmoset ESC-derived NS/PCs acquired similar gene expression profiles to those of fetal brain-derived NS/PCs by repeated passaging. Therefore, marmoset ESC-derived NS/PCs may be useful not only for accurate evaluation by allotransplantation of NS/PCs into non-human primate models, but are also applicable to analysis of iPSCs established from transgenic disease model marmosets.

Gene targeting and subsequent site-specific transgenesis at the β-actin (ACTB) locus in common marmoset embryonic stem cells.

Nonhuman primate embryonic stem (ES) cells have vast promise for preclinical studies. Genetic modification in nonhuman primate ES cells is an essential technique for maximizing the potential of these cells. The common marmoset (Callithrix jacchus), a nonhuman primate, is expected to be a useful transgenic model for preclinical studies. However, genetic modification in common marmoset ES (cmES) cells has not yet been adequately developed. To establish efficient and stable genetic modifications in cmES cells, we inserted the enhanced green fluorescent protein (EGFP) gene with heterotypic lox sites into the β-actin (ACTB) locus of the cmES cells using gene targeting. The resulting knock-in ES cells expressed EGFP ubiquitously under the control of the endogenous ACTB promoter. Using inserted heterotypic lox sites, we demonstrated Cre recombinase-mediated cassette exchange (RMCE) and successfully established a monomeric red fluorescent protein (mRFP) knock-in cmES cell line. Further, a herpes simplex virus-thymidine kinase (HSV-tk) knock-in cmES cell line was established using RMCE. The growth of tumor cells originating from the cell line was significantly suppressed by the administration of ganciclovir. Therefore, the HSV-tk/ganciclovir system is promising as a safeguard for stem cell therapy. The stable and ubiquitous expression of EGFP before RMCE enables cell fate to be tracked when the cells are transplanted into an animal. Moreover, the creation of a transgene acceptor locus for site-specific transgenesis will be a powerful tool, similar to the ROSA26 locus in mice.

Birth of common marmoset (Callithrix jacchus) offspring derived from in vitro-matured oocytes in chemically defined medium

Optimization of oocyte culture conditions is a crucial aspect of reproductive biology and technology. In the present study, maturation of germinal vesicle-stage marmoset oocytes were evaluated in the following media: Waymouth medium, Waymouth medium containing porcine follicular fluid (pFF) (Waymouth-pFF medium), and porcine oocyte medium (POM). Oocytes cultured in Waymouth-pFF medium had higher maturation rates to the metaphase II stage than those cultured in Waymouth medium (36.1% vs. 24.8%, respectively, P < 0.05), indicating the suitability of this medium for culturing marmoset oocytes. Hence, maturation of marmoset oocytes cultured in POM was subsequently evaluated. The rate of maturation to the metaphase I stage was significantly higher and degradation rates were significantly lower in oocytes cultured in POM than those cultured in Waymouth medium. In addition, three offspring were successfully obtained after transfer of embryos matured in chemically defined medium. Therefore, we concluded that POM was suitable for marmoset oocyte culture. Furthermore, this was apparently the first report of marmoset offspring derived from oocytes cultured in chemically defined medium.

Transgenic Monkey Model of the Polyglutamine Diseases Recapitulating Progressive Neurological Symptoms

Abstract Age-associated neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and the polyglutamine (polyQ) diseases, are becoming prevalent as a consequence of elongation of the human lifespan. Although various rodent models have been developed to study and overcome these diseases, they have limitations in their translational research utility owing to differences from humans in brain structure and function and in drug metabolism. Here, we generated a transgenic marmoset model of the polyQ diseases, showing progressive neurological symptoms including motor impairment. Seven transgenic marmosets were produced by lentiviral introduction of the human ataxin 3 gene with 120 CAG repeats encoding an expanded polyQ stretch. Although all offspring showed no neurological symptoms at birth, three marmosets with higher transgene expression developed neurological symptoms of varying degrees at 3–4 months after birth, followed by gradual decreases in body weight gain, spontaneous activity, and grip strength, indicating time-dependent disease progression. Pathological examinations revealed neurodegeneration and intranuclear polyQ protein inclusions accompanied by gliosis, which recapitulate the neuropathological features of polyQ disease patients. Consistent with neuronal loss in the cerebellum, brain MRI analyses in one living symptomatic marmoset detected enlargement of the fourth ventricle, which suggests cerebellar atrophy. Notably, successful germline transgene transmission was confirmed in the second-generation offspring derived from the symptomatic transgenic marmoset gamete. Because the accumulation of abnormal proteins is a shared pathomechanism among various neurodegenerative diseases, we suggest that this new marmoset model will contribute toward elucidating the pathomechanisms of and developing clinically applicable therapies for neurodegenerative diseases.

Development of rat tetraploid and chimeric embryos aggregated with diploid cells

In the present study, we examined the preimplantation and postimplantation development of rat tetraploid embryos produced by electrofusion of 2-cell-stage embryos. Developmental rate of tetraploid embryos to morula or blastocyst stage was 93% (56/60) and similar to that found in diploid embryos (95%, 55/58). After embryo transfer, rat tetraploid embryos showed implantation and survived until day 8 of pregnancy, however the conceptuses were aberrant on day 9. In mouse, tetraploid embryos have the ability to support the development of blastomeres that cannot develop independently. As shown in the present study, a pair of diploid blastomeres from the rat 8-cell-stage embryo degenerated immediately after implantation. Therefore, we examined whether rat tetraploid embryos have the ability to support the development of 2/8 blastomeres. We produced chimeric rat embryos in which a pair of diploid blastomeres from an 8-cell-stage green fluorescent protein negative (GFP-) embryo was aggregated with three tetraploid blastomeres from 4-cell GFP-positive (GFP+) embryos. The developmental rate of rat 2n(GFP-) <--> 4n(GFP+) embryos to the morula or blastocyst stages was 93% (109/117) and was similar to that found for 2n(GFP-) <--> 2n(GFP+) embryos (100%, 51/51). After embryo transfer, 2n(GFP-) <--> 4n(GFP+) conceptuses were examined on day 14 of pregnancy, the developmental rate to fetus was quite low (4%, 4/109) and they were all aberrant and smaller than 2n(GFP-) <--> 2n(GFP+) conceptuses, whereas immunohistochemical analysis showed no staining for GFP in fetuses. Our results suggest that rat tetraploid embryos are able to prolong the development of diploid blastomeres that cannot develop independently, although postimplantation development was incomplete.

Derivation of Induced Pluripotent Stem Cells by Retroviral Gene Transduction in Mammalian Species

Pluripotent stem cells can provide us with an enormous cell source for in vitro model systems for development. In 2006, new methodology was designed to generate pluripotent stem cells directly from somatic cells, and these cells were named induced pluripotent stem cells (iPSCs). This method consists of technically simple procedures: donor cell preparation, gene transduction, and isolation of embryonic stem cell-like colonies. The iPSC technology enables cell biologists not only to obtain pluripotent stem cells easily but also to study the reprogramming events themselves. Here, we describe the protocols to generate iPSCs from somatic origins by using conventional viral vectors. Specifically, we state the usage of three mammalian species: mouse, common marmoset, and human. As mouse iPSC donors, fibroblasts are easily prepared, while mesenchymal stem cells are expected to give rise to highly reprogrammed iPSCs efficiently. Common marmoset (Callithrix jacchus), a nonhuman primate, represents an alternative model to the usual laboratory animals. Finally, patient-specific human iPSCs give us an opportunity to examine the pathology and mechanisms of dysregulated genomic imprinting. The iPSC technology will serve as a valuable method for studying genomic imprinting, and conversely, the insights from these studies will offer valuable criteria to assess the potential of iPSCs.

Recent Progress in Reproductive Technologies based on the Common Marmoset (Callithrix Jacchus)

Abstract The common marmoset (Callithrix jacchus) is a non-endangered New World primate that is native to Brazil. Marmosets offer many advantages compared with other laboratory primates for studying reproductive biology: they are the only anthropoid primates that routinely ovulate multiple oocytes per ovarian cycle, have a short gestation period and reach sexual maturity at around 1 year of age. Moreover, it is possible to synchronize the ovarian cycle, and efficient protocols for superovulation have been developed over the last few decades. As this species is increasingly used in reproductive technology, basic technologies have been established to rival those available in Old World primates. In 2005, common marmoset embryonic stem (ES) cell lines were established and applied to several differentiation studies, which accelerated the development of regenerative therapies using human ES cells, and to the production of transgenic animals for human disease. With the recent development of induced pluripotent stem cells (iPS), non-human primate models using ES cells and iPS cells are needed for elucidation of the safety and efficacy of new technologies in regenerative medicine. In addition to their natural advantages as a model of humans, marmosets are also advantageous as experimental animals, and this should lead to a surge of interest among biological researchers.

Developing biomarkers for neurodegenerative diseases using genetically-modified common marmoset models

Mouse and non-human primate models of neurodegenerative disease: The prevalence of age-related neurodegenerative diseases continues to increase with ever increasing aging population over the age of 60. Although the difficulties associated with neurodegenerative diseases present an urgent global issue, there is no effective treatment for these conditions. To develop therapeutic methods and therapeutic agents for neurodegenerative diseases, model animals that simulate the human disease pathology are eagerly anticipated. There have been significant advancement in embryonic stem cell and genetic engineering in mice, and various transgenic models of neurodegenerative diseases provided great contribution to our understanding of basic disease mechanisms and the development of potential therapeutic molecules for neurodegenerative diseases (Rockenstein et al., 2007; Giuliani et al., 2017). However, differences between humans and rodents in the structure and physiological functions of the brain have resulted in difficulty in reproducing the selective vulnerability of specific neurons or circuits in mouse and rat models (Chan, 2013). Non-human primates, on the other hand, more closely share genetic, physiological, and morphological similarities with humans and can provide a better test system for drug and biomarker discovery for various psychological disorders and neurological diseases. Despite their value, non-human primates are not widely used due to limited availability of the animals, requiring a large breeding space, specialized breeders and veterans, which increases the cost of the study, not to mention the ethical issues. Among non-human primates, the common marmoset (Callithrix jacchus) is a small, non-endangered new world primate that is native to Brazil. Adult marmosets have an average height of 20–30 cm, weigh 350–450 g, and have advantages related to their small size. In comparison with macaque species, the relatively small size of marmosets translates to lower caging and feeding costs and reduced floor space requirements. Additionally, they can be handled relatively easily by one researcher. Marmosets also offer many advantages in studies of reproductive technology compared to other laboratory primates, such as a shorter gestation period, a faster sexual maturation, and higher fecundity, permitting the rapid establishment of gene-modified model lines. In fact, various basic research tools for marmosets have been developed recently, including transgenic (Sasaki et al., 2009) and genome-editing techniques (Sato et al., 2016) and elucidation of the entire genome sequence (Marmoset Genome Sequencing and Analysis Consortium, 2014). The natural advantages of the marmoset as a model of human systems in combination with its advantages as an experimental animal have led to a surge in interest among neuroscience researchers. In 2016, marmosets have been adopted for Brain/MINDS (Brain Mapping by Integrated Neurotechnologies for Disease Studies), a national brain project started in Japan with a goal to develop marmoset as a model animal for neuroscience. The project aims to: (1) build a multiscale marmoset brain map, (2) develop new technologies for researchers, and (3) create transgenic lines for modeling brain diseases. In this context, we focused on polyglutamine disease, a generic term for nine neurodegenerative diseases, including Huntington’s disease and various spinocerebellar ataxias, in an attempt to create an improved animal model of a human neurological disease. The abnormal elongation of the CAG repeat sequence encoding glutamine within the causative gene has revealed a common onset molecular mechanism that induces protein misfolding and aggregation, leading to neurodegeneration. Our first transgenic marmoset model for spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, used the cytomegalovirus (CMV) promoter to induce transgene expression and progressive neurological symptoms, including motor impairment (Tomioka et al., 2017b). Pathological examination revealed neurodegeneration and intranuclear polyglutamine protein inclusions accompanied by gliosis, all of which recapitulate the neuropathological features of patients with polyglutamine disease. Consistent with the neuronal loss in the cerebellum, non-invasive brain magnetic resonance imaging of a living symptomatic marmoset showed cerebellar atrophy. However, the model had a high fetal death rate, widespread juvenile disease onset, and rapid disease progression, which are unusual in humans with SCA3. Moreover, F1 offspring were difficult to obtain by natural mating. It appears that, although ubiquitously expressed promoters induce strong transgene expression, they also lead to irrelevant symptoms and abnormalities in fetal development. Therefore, controlling transgene expression is one of the most important challenges in the development of more sophisticated animal models of human disease.