Attempts for deriving extended pluripotent stem cells from common marmoset embryonic stem cells.

Extended pluripotent stem cells (EPSCs) derived from mice and humans showed an enhanced potential for chimeric formation. By exploiting transcriptomic approaches, we assessed the differences in gene expression profile between extended EPSCs derived from mice and humans, and those newly derived from the common marmoset (marmoset; Callithrix jacchus). Although the marmoset EPSC-like cells displayed a unique colony morphology distinct from murine and human EPSCs, they displayed a pluripotent state akin to embryonic stem cells (ESCs), as confirmed by gene expression and immunocytochemical analyses of pluripotency markers and three-germ-layer differentiation assay. Importantly, the marmoset EPSC-like cells showed interspecies chimeric contribution to mouse embryos, such as E6.5 blastocysts in vitro and E6.5 epiblasts in vivo in mouse development. Also, we discovered that the perturbation of gene expression of the marmoset EPSC-like cells from the original ESCs resembled that of human EPSCs. Taken together, our multiple analyses evaluated the efficacy of the method for the derivation of marmoset EPSCs.

Multimodal analyses of a non-human primate model harboring mutant amyloid precursor protein transgenes driven by the human EF1α promoter.

Alzheimer's disease (AD) is the leading cause of dementia which afflicts tens of millions of people worldwide. Despite many scientific progresses to dissect the AD's molecular basis from studies on various mouse models, it has been suffered from evolutionary species differences. Here, we report generation of a non-human primate (NHP), common marmoset model ubiquitously expressing Amyloid-beta precursor protein (APP) transgenes with the Swedish (KM670/671NL) and Indiana (V717F) mutations. The transgene integration of generated two transgenic marmosets (TG1&TG2) was thoroughly investigated by genomic PCR, whole-genome sequencing, and fluorescence in situ hybridization. By reprogramming, we confirmed the validity of transgene expression in induced neurons in vitro. Moreover, we discovered structural changes in specific brain regions of transgenic marmosets by magnetic resonance imaging analysis, including in the entorhinal cortex and hippocampus. In immunohistochemistry, we detected increased Aß plaque-like structures in TG1 brain at 7 years old, although evident neuronal loss or glial inflammation was not observed. Thus, this study summarizes our attempt to establish an NHP AD model. Although the transgenesis approach alone seemed not sufficient to fully recapitulate AD in NHPs, it may be beneficial for drug development and further disease modeling by combination with other genetically engineered models and disease-inducing approaches.

Homologous Recombination-Enhancing Factors Identified by Comparative Transcriptomic Analyses of Pluripotent Stem Cell of Human and Common Marmoset.

A previous study assessing the efficiency of the genome editing technology CRISPR-Cas9 for knock-in gene targeting in common marmoset (marmoset; Callithrix jacchus) embryonic stem cells (ESCs) unexpectedly identified innately enhanced homologous recombination activity in marmoset ESCs. Here, we compared gene expression in marmoset and human pluripotent stem cells using transcriptomic and quantitative PCR analyses and found that five HR-related genes (BRCA1, BRCA2, RAD51C, RAD51D, and RAD51) were upregulated in marmoset cells. A total of four of these upregulated genes enhanced HR efficiency with CRISPR-Cas9 in human pluripotent stem cells. Thus, the present study provides a novel insight into species-specific mechanisms for the choice of DNA repair pathways.

Establishment of an induced pluripotent stem cell line from a female domestic ferret (Mustela putorius furo) with an X chromosome instability.

The domestic ferret (ferret; Mustela putorius furo) is an important animal model for neuroscience and preclinical/veterinary medicine owing to its highly developed cerebral cortex and susceptibility to avian influenza and corona viruses. Nevertheless, there is a lack of in vitro ferret models, since immortal cell lines including induced pluripotent stem cells (iPSCs) of ferrets have been scarce. In this study, we established an iPSC line from ferret skin fibroblasts. The established iPSC line, fiPS-1, showed standard characteristics of pluripotency, but its X chromosome was unstable. Collectively, the present study provides a useful resource for in vitro model using the ferret.

Non-viral Induction of Transgene-free iPSCs from Somatic Fibroblasts of Multiple Mammalian Species.

Induced pluripotent stem cells (iPSCs) are capable of providing an unlimited source of cells from all three germ layers and germ cells. The derivation and usage of iPSCs from various animal models may facilitate stem cell-based therapy, gene-modified animal production, and evolutionary studies assessing interspecies differences. However, there is a lack of species-wide methods for deriving iPSCs, in particular by means of non-viral and non-transgene-integrating (NTI) approaches. Here, we demonstrate the iPSC derivation from somatic fibroblasts of multiple mammalian species from three different taxonomic orders, including the common marmoset (Callithrix jacchus) in Primates, the dog (Canis lupus familiaris) in Carnivora, and the pig (Sus scrofa) in Cetartiodactyla, by combinatorial usage of chemical compounds and NTI episomal vectors. Interestingly, the fibroblasts temporarily acquired a neural stem cell-like state during the reprogramming. Collectively, our method, robustly applicable to various species, holds a great potential for facilitating stem cell-based research using various animals in Mammalia.

Primed to Naive-Like Conversion of the Common Marmoset Embryonic Stem Cells.

Mammalian pluripotent stem cells are thought to exist in two states: naive and primed. Generally, unlike those in rodents, pluripotent stem cells in primates, including humans, are regarded as being in the primed pluripotent state. Recently, several groups reported the existence of naive pluripotent stem cells in humans. In this study, we report the conversion of primed state embryonic stem cells from common marmoset, a New World monkey, to the naive state using transgenes. The cells showed typical naive state features, including dome-like colony morphology, growth factor requirement, gene expression profile, X chromosome activation state, and energy metabolic status. Moreover, interspecies chimeric embryo formation ability with mouse embryos was increased in the naive state. This technique can be applied in basic medical research using nonhuman primates, such as preclinical use of naive pluripotent stem cells and generating genetically modified primates.

Generation of a male common marmoset embryonic stem cell line DSY127-BV8VT1 carrying double reporters specific for the germ cell linage using the CRISPR-Cas9 and PiggyBac transposase systems.

BLIMP1 (PRDM1) and VASA (DDX4) play pivotal roles in the development of the germ cell linage. Importantly, these genes are specifically expressed in germ cells; BLIMP1 in primordial germ cells (PGCs) to early-stage gonocytes, and VASA in migration-stage PGCs to mature gametes. The high reproductive efficiency of common marmosets (marmosets; Callithrix jacchus) makes them advantageous for use in germ cell research. We herein report the generation of a male marmoset embryonic stem cell (ESC) line harboring BLIMP1 and DDX4 double reporters. This ESC line will be a useful tool for investigating male gametogenesis in non-human primates.

Evaluating the efficacy of small molecules for neural differentiation of common marmoset ESCs and iPSCs.

The common marmoset (marmoset; Callithrix jacchus) harbors various desired features as a non-human primate (NHP) model for neuroscience research. Recently, efforts have been made to induce neural cells in vitro from marmoset pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), which are characterized by their capacity to differentiate into all cell types from the three germ layers. Successful generation of marmoset neural cells is not only invaluable for understanding neural development and for modeling neurodegenerative and psychiatric disorders, but is also necessary for the phenotypic screening of genetically-modified marmosets. However, differences in the differentiation propensity among PSC lines hamper the applicability and the reproducibility of differentiation methods. To overcome this limitation, we evaluated the efficacy of small molecules for neural differentiation of marmoset ESCs (cjESCs) and iPSCs using multiple differentiation methods. By immunochemical and transcriptomic analyses, we confirmed that our methods using the small molecules are efficient for various differentiation protocols by either enhancing the yield of a mixture of neural cells including both neurons and glial cells, or a pure population of neurons. Collectively, our findings optimized in vitro neural differentiation methods for marmoset PSCs, which would ultimately help enhance the utility of the animal model in neuroscience.

A versatile toolbox for knock-in gene targeting based on the Multisite Gateway technology.

Knock-in (KI) gene targeting can be employed for a wide range of applications in stem cell research. However, vectors for KI require multiple complicated processes for construction, including multiple times of digestion/ligation steps and extensive restriction mapping, which has imposed limitations for the robust applicability of KI gene targeting. To circumvent this issue, here we introduce versatile and systematic methods for generating KI vectors by molecular cloning. In this approach, we employed the Multisite Gateway technology, an efficient in vitro DNA recombination system using proprietary sequences and enzymes. KI vector construction exploiting these methods requires only efficient steps, such as PCR and recombination, enabling robust KI gene targeting. We show that combinatorial usage of the KI vectors generated using this method and site-specific nucleases enabled the precise integration of fluorescent protein genes in multiple loci of human and common marmoset (marmoset; Callithrix jacchus) pluripotent stem cells. The methods described here will facilitate the usage of KI technology and ultimately help to accelerate stem cell research.

Establishment of induced pluripotent stem cells from common marmoset fibroblasts by RNA-based reprogramming.

The common marmoset (marmoset; Callithrix jacchus) shows anatomical and physiological features that are in common with humans. Establishing induced pluripotent stem cells (iPSCs) from marmosets holds promise for enhancing the utility of the animal model for biomedical and preclinical studies. However, in spite of the presence of some previous reports on marmoset iPSCs, the reprogramming technology in marmosets is still under development. In particular, the efficacy of RNA-based reprogramming has not been thoroughly investigated. In this study, we attempted RNA-based reprogramming for deriving iPSCs from marmoset fibroblasts. Although we failed to derive iPSC colonies from marmoset fibroblasts by using a conventional RNA-based reprogramming method previously validated in human fibroblasts, we succeeded in deriving colony-forming cells with a modified induction medium supplemented with a novel set of small molecules. Importantly, following one-week culture of the colony-forming cells in conventional embryonic stem cell (ESC) medium, we obtained iPSCs which express endogenous pluripotent markers and show a differentiation potential into all three germ layers. Taken together, our results indicate that RNA-based reprogramming, which is valuable for deriving transgene-free iPSCs, is applicable to marmosets.

Development of Non-proximate Probe Electrospray Ionization for Real-Time Analysis of Living Animal.

Ambient ionization mass spectrometry is one of the most challenging analytical tools in the field of biomedical research. We previously demonstrated that probe electrospray ionization mass spectrometry (PESI-MS) could potentially be used in the rapid diagnosis of cancer. Although this technique does not require a tedious sample pretreatment process, it was not possible for our previously reported setup to be applied to cases involving the direct sampling of tissues from living animal and large animal subjects, because there would not be enough room to accommodate the larger bodies juxtaposed to the ion inlet. To make PESI-MS more applicable for the real-time analysis of living animals, a long auxiliary ion sampling tube has been connected to the ion inlet of the mass spectrometer to allow for the collection of ions and charged droplets from the PESI source (hereafter, referred to as non-proximate PESI). Furthermore, an additional ion sampling tube was connected to a small diaphragm pump to increase the uptake rate of air carrying the ions and charged droplets to the ion inlet. This modification allows for the extended ion sampling orifice to be positioned closer to the specimens, even when they are too large to be placed inside the ionization chamber. In this study, we have demonstrated the use of non-proximate PESI-MS for the real-time analysis for biological molecules and pharmacokinetic parameters from living animals.