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Phosphorus is an essential substance in our body, and hypophosphataemia (HP) is well-described in rickets, refeeding syndrome, diabetic ketoacidosis (DKA), and in chronic alcohol-abuse. However, to our knowledge, HP among severely-malnourished children has not been studied in detail, and information on prevalence, severity, and treatment is scarce. Currently, there are only a few published case reports of HP. This case series describes three cases of HP that presented to Dhaka Hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b). Our first case required mechanical ventilation for respiratory distress associated with severe hypokalaemia (K 1.1 mmol/L) and moderate hypophosphataemia (P 2.1 mg/dL). The second case presented with severe sepsis which was associated with symptomatic hypocalcaemia (Ca 1.68 mmol/L), hypokalaemia (K 1.82 mmol/L), and severe hypophosphataemia (P 0.9 mg/dL). The third case presented with pneumonia and sepsis which were complicated by hypokalaemia (K 2.05 mmol/L) and severe hypophosphataemia (P 1.1 mg/dL). Marked lethargy and severe hypotonia were associated with HP in all of these cases. Manifestations of HP are diverse and can occur in association with other electrolyte imbalances, especially among malnourished children. Malnutrition, combined with sepsis, is one of the major killers of children younger than 5 years of age, and both malnutrition and sepsis can cause HP. It is concluded that the underlying causes of morbidity, including HP, should be actively sought and treated to reduce the mortality of children aged below five years. [ABSTRACT FROM AUTHOR]

Yoshimatsu S, Ando Y, Terazaki H, Sakashita N, Tada S, Yamashita T, Suga M, Uchino M, Ando M (Kumamoto University School of Medicine; and Saiseikai Hospital, Kumamoto, Japan). Endoscopic and pathological manifestations of the gastrointestinal tract in familial amyloidotic polyneuropathy type I (Met30). J Intern Med 1998; 243: 65–72. ObjectivesTo evaluate the characteristic changes in the gastrointestinal tract in familial amyloidotic polyneuropathy (FAP) (Met30), both fibre gastroscopy and colonoscopy studies were performed in FAP (Met30) patients. Microscopic changes were also examined in autopsied and biopsied materials from patients with FAP, and compared with data from autopsied samples from patients with AL amyloidosis, and secondary amyloidosis patients. DesignEndoscopic and histopathological study. SettingKumamoto University Hospital, Kumamoto, Japan. SubjectsNine patients with FAP (Met30) underwent fibre gastroscopy and colonoscopy. Six autopsied and 23 biopsied gastrointestinal samples from FAP patients, four from autopsied amyloidosis (including two myeloma associated form), and two from autopsied secondary amyloidosis patients were examined for histopathological study. Main outcome measuresFibre gastroscopy and colonoscopy were employed for macroscopic study. Congo red and H-E staining were performed for histopathological study. Macroscopic changes in the gastrointestinal tract and microscopic differences in the amyloid distribution pattern were compared between the different types of amyloidosis. ResultsFibre gastroscopy and colonoscopy for nine FAP patients revealed that four showed a fine granular appearance in the duodenum, three showed lack of lustre, and two showed mucosal friability in the gastrointestinal tract; however, no macroscopic abnormality was observed in four other FAP patients. Histopathological examination of tissue from FAP patients revealed that, although a small amount of amyloid was recognized in the submucosa perivascular layer, a significant amount of amyloid was seen in and around the nerves of the gastrointestinal tract, but very little in Auerbach's nerve plexus. In total, the amount of deposited amyloid in the tissues was small compared with that in other types of systemic amyloidosis, such as AL and secondary amyloidosis. ConclusionThese results suggest that the major reason why FAP patients show such severe gastrointestinal symptoms, compared with other types of systemic amyloidosis, may be because of the deposition of a significant amount of amyloid in the nerves in the gastrointestinal tract. [ABSTRACT FROM AUTHOR]

Purpose: To analyze the influence of liver dysfunction and parenchymal pathology on the accumulation of superparamagnetic iron oxide (SPIO).Methods: We evaluated MR images of 13 patients having small hepatic neoplasms before and after administration of SPIO (10 micronol/kg). Biopsy and laboratory data confirmed the presence of severe cirrhosis in two patients, mild cirrhosis in four, chronic hepatitis in five, and normal livers in two. Degrees of liver dysfunction or liver parenchymal pathology were correlated with reductions in signal intensity of the liver and spleen after administration of SPIO. Signal intensity reduction was evaluated using a 1.5 Tesla MR unit.Results: Response to SPIO of the liver and spleen did not correlate with liver parenchymal pathology, although reductions in signal intensity of the liver were somewhat small in severely cirrhotic livers. There were slight correlations between signal intensity alterations of the liver and laboratory data such as the indocyanine green retention rate (correlation coefficient 0. 47), albumin (0.36), total bilirubin (0.36), and serum glutamic oxaloacetic transaminase (GOT) (0.46). Signal intensity reduction of spleen did not correlate with liver function tests except for serum GOT. In patients with cirrhosis, heterogeneous structures were detected in the nontumorous portions of the liver. However, these did not prevent the diagnosis of small hepatomas.Conclusion: The uptake of SPIO showed some correlation with liver function but not with chronic liver parenchymal pathology. SPIO provided sufficient contrast between tumor and surrounding liver parenchyma among patients with chronic liver disease. [ABSTRACT FROM AUTHOR]

We isolated 12 polymorphic microsatellites from the noxious red-tide-causing alga Chattonella ovata. These loci provide a class of highly variable genetic markers, as the number of alleles ranged from four to 12, and the observed and expected heterozygosities ranged from 0.238 to 0.850 and from 0.310 to 0.889, respectively. These loci are useful for revealing the genetic structure of and gene flow among C. ovata populations. [ABSTRACT FROM AUTHOR]

The generation of reactive free radicals by Chattonella antiqua ('Red Tide'), which may be a causative factor in fish death, has been demonstrated by electron spin resonance spectroscopy and a microelectrode technique. Electrochemical detection indicates that superoxide anions are produced as a primary product, and undergo further reactions during diffusion in glycocalyx to produce hydrogen, hydroxy-, and carbon-centered radicals which are detected by ESR as secondary products. It is proposed that these reactive radicals are responsible for the destruction of the mucous membranes of fish on exposure to 'Red Tide'. [ABSTRACT FROM AUTHOR]

Non-human primates (NHPs) are the closest animal model to humans; thus, gene engineering technology in these species holds great promise for the elucidation of higher brain functions and human disease models. Knockin (KI) gene targeting is a versatile approach to modify gene(s) of interest; however, it generally suffers from the low efficiency of homology-directed repair (HDR) in mammalian cells, especially in non-expressed gene loci. In the current study, we generated a tyrosine hydroxylase (TH)-2A-Cre KI model of the common marmoset monkey (marmoset; Callithrix jacchus) using an HDR-biased CRISPR-Cas9 genome editing approach using Cas9-DN1S and RAD51. This model should enable labeling and modification of a specific neuronal lineage using the Cre-loxP system. Collectively, the current study paves the way for versatile gene engineering in NHPs, which may be a significant step toward further biomedical and preclinical applications., Competing Interests: Declaration of interests H.O. has been a paid scientific advisory board member of San Bio Co. Ltd., Regenerative Medicine iPS Gateway Center Co. Ltd., and K Pharma, Inc. S.Y. has been a paid associate researcher of Daiichi-Sankyo RD Novare Co. Ltd. However, there was no effect of these companies on the interpretation, writing, or publication of this study. H.O. and S.Y. declare that there are no non-financial conflicts of interest with this work. In addition, the other authors declare that there are neither financial nor non-financial conflicts of interest., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: To examine the fracture strength and fracture mode of flared root canals reinforced with different post and core materials., Materials and Methods: Forty endodontically treated bovine teeth structured to mimic human mandibular premolars with flared root canals were reinforced with resin composite and glass fiber post (FRC), composite resin (RC), ceramic core (LD), and ceramic core with resin composite reinforcement (RLD), and restored with single zirconia crowns (n = 10 in each group). The fracture strength and mode of the root canals restored with zirconia crown were assessed. The fracture strength was compared with a one-way analysis of variance (ANOVA) following Tukey HSD tests. A multiple regression analysis was conducted to test the effect of the post/core materials on the fracture loads. Fisher's exact test was used in the failure mode analysis., Results: The mean fracture strength of RLD was significantly higher than RC, FRC, and LD (p < 0.05), while no significant differences were found among RC, FRC, and LD (p < 0.05). The regression analysis found that the fracture strength using the lithium disilicate was significantly lower for the post and higher for the core than that using the resin composite (p < 0.05), and there were no significant difference in the fracture strengths between the resin composite and glass fiber used for the post (p > 0.05). Most of the specimens exhibited root fractures, and no significant differences were observed among the groups (p < 0.05)., Conclusions: The results of this study suggest that reinforcement of flared root canals using a combination of resin composite for the core and lithium disilicate ceramic for the post is superior to resin composite and glass fiber in mechanical properties when restoring a single crown., (© 2022 by the American College of Prosthodontists.)

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is an inherited cerebral small vessel disease (CSVD) caused by biallelic mutations in the high-temperature requirement serine peptidase A1 (HTRA1) gene. Even heterozygous mutations in HTRA1 are recently revealed to cause cardinal clinical features of CSVD. Here, we report the first establishment of a human induced pluripotent stem cell (hiPSC) line from a patient with heterozygous HTRA1-related CSVD. Peripheral blood mononuclear cells (PBMCs) were reprogrammed by the transfection of episomal vectors encoding human OCT3/4 (POU5F1), SOX2, KLF4, L-MYC, LIN28, and a murine dominant-negative mutant of p53 (mp53DD). The established iPSCs had normal morphology as human pluripotent stem cells and normal karyotype (46XX). Moreover, we found that the HTRA1 missense mutation (c.905G>A, p.R302Q) was heterozygous. These iPSCs expressed pluripotency-related markers and had the potential to differentiate into all three germ layers in vitro. HTRA1 and the supposed disease-associated gene NOG were differentially expressed in the patient iPSCs at mRNA levels compared to those of control lines. The iPSC line would facilitate in vitro research for understanding the cellular pathomechanisms caused by the HTRA1 mutation including its dominant-negative effect., (© 2023. The Author(s).)

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., (© 2022 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Purpose: This study aimed to evaluate the surface strain at the cervical area of endodontically treated molars with a large pulp chamber restored using a composite resin core with three different types of core build-up systems., Methods: Reproduction models of human mandibular molars with prepared post spaces were used in this study. Roots duplicated with a composite resin were used as the experimental teeth. Three types of core build-up systems were used: composite resin core(RC), composite resin core with fiber posts (FC), and composite resin core with a prefabricated zirconia tube (ZC). Each group comprised eight specimens. Crowns made of yttria partially stabilized zirconia were cemented with dual-cure resin cement. Four strain gauges were attached to the surfaces of each specimen: the cervical area of the root and crown, on the buccal and lingual sides. The surface strain at each cervical area was measured using a static loading test and statistically analyzed., Results: In the case of static loading to the buccal cusp inner slope, ZC showed a significantly lower strain than RC in the crown on the buccal side and in the root and FC in the root. In the central fossa, ZC showed a significantly lower strain than FC in the root on the lingual side., Conclusions: The prefabricated zirconia tube reduced the surface strain at the cervical area of the buccal/lingual root in molars; however, the effect was small in the cervical area of the crown.

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 Aβ 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., (Copyright © 2022. Published by Elsevier B.V.)

Objectives: Endoscopic submucosal resection with band ligation (ESMR-L) and endoscopic submucosal dissection (ESD) are both standard endoscopic resection methods for rectal neuroendocrine tumors (NETs) <10 mm in size. However, there is no definitive consensus on which is better. Here, we compared the efficacy of ESMR-L and ESD for small rectal NETs., Methods: This was a multicenter retrospective cohort study including 205 patients with rectal NETs who underwent ESMR-L or ESD. Treatment outcomes were compared by univariate analysis, multivariate analysis, and inverse probability treatment weighting (IPTW) using propensity scores. Subgroup analysis evaluated the impact of the endoscopist's experience on the technical outcome., Results: Eighty-nine patients were treated by ESMR-L and 116 by ESD. The R0 resection rate was not significantly different between the two (90% vs. 92%, p = 0.73). The procedure time of ESMR-L was significantly shorter than for ESD (17 min vs. 52 min, p < 0.01) and the hospitalization period was also significantly shorter (3 days vs. 5 days, p < 0.01). These results were confirmed by multivariate analysis and also after IPTW adjustment. The procedure time of ESD was significantly prolonged by a less-experienced endoscopist (49 min vs. 70 min, p = 0.02), but that of ESMR-L was not affected (17 min vs. 17 min, p = 0.27)., Conclusions: For small rectal NETs, both ESMR-L and ESD showed similar high complete resection rates. However, considering the shorter procedure time and shorter hospitalization period, ESMR-L is the more efficient treatment method, especially for less-experienced endoscopists., Competing Interests: None., (© 2022 The Authors. DEN Open published by John Wiley & Sons Australia, Ltd on behalf of Japan Gastroenterological Endoscopy Society.)

In developed countries, the aging of the population and the associated increase in age-related diseases are causing major unresolved medical, social, and environmental matters. Therefore, research on aging has become one of the most important and urgent issues in life sciences. If the molecular mechanisms of the onset and progression of neurodegenerative diseases are elucidated, we can expect to develop disease-modifying methods to prevent neurodegeneration itself. Since the discovery of induced pluripotent stem cells (iPSCs), there has been an explosion of disease models using disease-specific iPSCs derived from patient-derived somatic cells. By inducing the differentiation of iPSCs into neurons, disease models that reflect the patient-derived pathology can be reproduced in culture dishes, and are playing an active role in elucidating new pathological mechanisms and as a platform for new drug discovery. At the same time, however, we are faced with a new problem: how to recapitulate aging in culture dishes. It has been pointed out that cells differentiated from pluripotent stem cells are juvenile, retain embryonic traits, and may not be fully mature. Therefore, attempts are being made to induce cell maturation, senescence, and stress signals through culture conditions. It has also been reported that direct conversion of fibroblasts into neurons can reproduce human neurons with an aged phenotype. Here, we outline some state-of-the-art insights into models of neuronal aging in vitro . New frontiers in which stem cells and methods for inducing differentiation of tissue regeneration can be applied to aging research are just now approaching, and we need to keep a close eye on them. These models are forefront and intended to advance our knowledge of the molecular mechanisms of aging and contribute to the development of novel therapies for human neurodegenerative diseases associated with aging., Competing Interests: HO was a founder scientist and a Scientific Advisory Board member for SanBio Co., Ltd., and K Pharma Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Inagaki, Yoshimatsu and Okano.)

Potentials of immortal proliferation and unlimited differentiation into all the three germ layers and germ cells in induced pluripotent stem cells (iPSCs) render them important bioresources for in vitro reconstitution and modeling of intravital tissues and organs in various animal models, thus contributing to the elucidation of pathomechanisms, drug discovery and stem cell-based regenerative medicine. We previously reported promising approaches for deriving transgene-free iPSCs from somatic fibroblasts of multiple mammalian species by episomal vector or RNA transfection, although the respective step-by-step protocols and the combinatorial usage of these methods, which achieved high induction efficiency, have not been described in the literature so far. Here, we provide a detailed step-by-step description of these methods with critical tips and slight modifications (improvements) to previously reported methods. We also report a novel method for the establishment of iPSCs from the Syrian hamster (also known as golden hamster; Mesocricetus auratus), a unique animal model of hibernation. We anticipate this methodology will contribute to stem cell biology and regenerative medicine research., (© 2022 Japanese Society of Developmental Biologists.)

This case report outlines a clinically undetected urinary bladder plasmacytoid urothelial carcinoma (PUC) with multiple metastases detected at autopsy. An 89-year-old man presented with edema in the lower limbs. Pleural fluid cytology revealed discohesive carcinomatous cells, although imaging studies failed to identify the primary site of tumor. The patient died of respiratory failure. Autopsy disclosed a prostate tumor and diffusely thickened urinary bladder and rectum without distinct tumorous lesions. Histologically, the tumor consisted of acinar-type prostate adenocarcinoma with no signs of metastasis. Additionally, small, plasmacytoid tumor cells were observed in the urinary bladder/rectum as isolated or small clustering fashions. These metastasized to the lungs, intestine, generalized lymph nodes in a non-mass-forming manner. Combined with immunohistochemical studies, these tumor cells were diagnosed PUC derived from the urinary bladder. Both clinicians and pathologists should recognize PUC as an aggressive histological variant, which can represent a rapid systemic progression without mass-forming lesions.

Background: Microglia are innate immune cells that are the only residential macrophages in the central nervous system. They play vital physiological roles in the adult brain and during development. Microglia are particularly in the spotlight because many genetic risk factors recently identified for neurodegenerative diseases are largely expressed in microglia. Rare polymorphisms in these risk alleles lead to abnormal activity of microglia under traumatic or disease conditions., Methods: In the present study, to investigate the multifaceted functions of human microglia, we established a novel robust protocol to generate microglia from human induced pluripotent stem cells (hiPSCs) using a combination of cytokines and small chemicals essential for microglia ontogeny. Moreover, we highly enhanced the microglial differentiation efficiency by forcing the expression of PU.1, a crucial transcription factor for microglial development, during posterior mesoderm differentiation., Results: By our novel method, we demonstrated the generation of a greater number of hiPSC-derived microglia (hiMGLs, approximately 120-folds) than the prior methods (at most 40-folds). Over 90% of the hiMGLs expressed microglia-specific markers, such as CX3CR1 and IBA-1. Whole-transcriptome analysis revealed that these hiMGLs are similar to human primary microglia but differ from monocytes/macrophages. Furthermore, the specific physiological functions of microglia were confirmed through indices of lipopolysaccharide responsiveness, phagocytotic ability, and inflammasome formation. By co-culturing these hiMGLs with mouse primary neurons, we demonstrated that hiMGLs can regulate the activity and maturation of neurons., Conclusions: In this study, our new simple, rapid, and highly efficient method for generating microglia from hiPSCs will prove useful for future investigations on microglia in both physiological and disease conditions, as well as for drug discovery., (© 2022. The Author(s).)

Recent success in derivation of functional gametes (oocytes and spermatozoa) from pluripotent stem cells (PSCs) of rodents has made it feasible for future application to large animals including endangered species and to ultimately humans. Here, we summarize backgrounds and recent studies on in vitro gametogenesis from rodent PSCs, and similar approaches using PSCs from large animals, including livestock, nonhuman primates (NHPs), and humans. We also describe additional developing approaches for in vitro reconstitution of reproductive organs, such as the ovary (ovarioid), testis (testisoid), and future challenges in the uterus (uteroid) and oviduct (oviductoid), all of which may be derived from PSCs. Once established, these in vitro systems may serve as a robust platform for elucidating the pathology of infertility-related disorders and ectopic pregnancy, principle of reproduction, and artificial biogenesis. Therefore, these possibilities, especially when using human cells, require consideration of ethical issues, and international agreements and guidelines need to be raised before opening "Pandora's Box".

Introduction: Efficient induction of the otic placode, the developmental origin of the inner ear from human pluripotent stem cells (hPSCs), provides a robust platform for otic development and sensorineural hearing loss modelling. Nevertheless, there remains a limited capacity of otic lineage specification from hPSCs by stepwise differentiation methods, since the critical factors for successful otic cell differentiation have not been thoroughly investigated. In this study, we developed a novel differentiation system involving the use of a three-dimensional (3D) floating culture with signalling factors for generating otic cell lineages via stepwise differentiation of hPSCs., Methods: We differentiated hPSCs into preplacodal cells under a two-dimensional (2D) monolayer culture. Then, we transferred the induced preplacodal cells into a 3D floating culture under the control of the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), retinoic acid (RA) and WNT signalling pathways. We evaluated the characteristics of the induced cells using immunocytochemistry, quantitative PCR (qPCR), population averaging, and single-cell RNA-seq (RNA-seq) analysis. We further investigated the methods for differentiating otic progenitors towards hair cells by overexpression of defined transcription factors., Results: We demonstrated that hPSC-derived preplacodal cells acquired the potential to differentiate into posterior placodal cells in 3D floating culture with FGF2 and RA. Subsequent activation of WNT signalling induced otic placodal cell formation. By single-cell RNA-seq (scRNA-seq) analysis, we identified multiple clusters of otic placode- and otocyst marker-positive cells in the induced spheres. Moreover, the induced otic cells showed the potential to generate hair cell-like cells by overexpression of the transcription factors ATOH1, POU4F3 and GFI1 ., Conclusions: We demonstrated the critical role of FGF2, RA and WNT signalling in a 3D environment for the in vitro differentiation of otic lineage cells from hPSCs. The induced otic cells had the capacity to differentiate into inner ear hair cells with stereociliary bundles and tip link-like structures. The protocol will be useful for in vitro disease modelling of sensorineural hearing loss and human inner ear development and thus contribute to drug screening and stem cell-based regenerative medicine., Competing Interests: H.O. is a founding scientist and scientific advisor of SanBio Co. Ltd. and K Pharma Inc. M.H., K.O. and M.F. are co-founders of Otolink Inc. The other authors indicate no potential conflicts of interest., (© 2022 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

Background: Fine-tuned cochlear development is essential for hearing. Owing to the difficulty in using early human fetal samples, most of our knowledge regarding cochlear development has been obtained from rodents. However, several inter-species differences in cochlear development between rodents and humans have been reported. To bridge these differences, we investigated early otic development of a non-human primate model animal, the common marmoset (Callithrix jacchus)., Methods: We examined 20 genes involved in early cochlear development and described the critical developmental steps for morphogenesis, which have been reported to vary between rodents and marmosets., Results: The results revealed that several critical genes involved in prosensory epithelium specifications showed higher inter-species differences, suggesting that the molecular process for hair cell lineage acquisition in primates differs considerably from that of rodents. We also observed that the tempo of cochlear development was three times slower in the primate than in rodents., Conclusions: Our data provide new insights into early cochlear development in primates and humans and imply that the procedures used for manipulating rodent cochlear sensory cells cannot be directly used for the research of primate cells due to the intrinsic inter-species differences in the cell fate determination program., (© 2022. The Author(s).)

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.

The defective and persistent Sendai virus (SeVdp) vector system allows efficient generation of transgene-free induced pluripotent stem cells (iPSCs) from human somatic cells. By leveraging the system, here we report the generation of an iPSC line from somatic fibroblasts of a healthy control donner (female), named KEIOi002-A (also named YG-iPS). The control iPSC line would be a useful resource for stem cell research and regenerative medicine., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Epstein-Barr virus (EBV)-based episomal vector system enables persistent transgene expression, which is advantageous for efficient derivation of transgene-free induced pluripotent stem cells (iPSCs) without viral transduction. Here, we report establishment of an iPSC line from somatic fibroblasts of a neonatal common marmoset monkey (marmoset; Callithrix jacchus) using an all-in-one episomal vector that we newly developed. The established iPSC line, named NM-iPS, showed standard characteristics of pluripotency such as pluripotency-related marker expression, three germ layer differentiation, and normal karyotype (2n = 46). The novel iPSC line would be a useful resource for stem cell research using non-human primates., (Published by Elsevier B.V.)

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., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

We previously reported the non-viral derivation of transgene-free induced pluripotent stem cells (iPSCs) from somatic fibroblasts of a female beagle dog using an optimized induction medium and integration-free episomal vectors. Here, we report novel derivation of a male canine iPSC line OF35Y-iPS, which showed standard characteristics of pluripotency such as a strong gene expression profile of pluripotency markers, differentiation potential into all three germ layers, and normal karyotype (78XY). Furthermore, we demonstrated targeted integration of 2A-EGFP into the canine NANOS3 locus. The novel iPSC line would be a useful resource for stem cell research and regenerative veterinary medicine., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

POU class 5 homeobox 1 (POU5F1, also known as OCT4) is critical for maintenance of pluripotency, germ cell fate, reprogramming into a pluripotent state, and early embryogenesis. We generated an embryonic stem cell (ESC) line of the common marmoset (Callithrix jacchus) harboring a heterozygous knock-in allele of OCT4-P2A-mCerulean-T2A-pac. The ESC line (CMES40-OC) will be valuable for investigation of primed/naïve pluripotency and germ cell fate. Homozygous OCT4 knock-in clones were generated but could not be sustained in an undifferentiated state in long-term culture. The OCT4 knock-in system facilitated simultaneous knock-in of a reporter construct at another locus, DDX4 (VASA)., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

We previously reported the efficient targeted introduction of transgenes into the genomic DNA of the common marmoset (Callithrix jacchus) using CRISPR-Cas9. In this study, we generated a marmoset embryonic stem cell (ESC) line that ubiquitously expresses the tamoxifen-inducible Cre-driver ERT2CreERT2. We validated the pluripotency of the ESC line and also successfully demonstrated the temporal control of the Cre-driver in a tamoxifen-dependent manner in the ESCs. This ESC line, named ActiCre-B1, will be a valuable resource for in vitro investigation of phenotypes related to embryonic lethality by targeted knockout of functionally important genes., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

The common marmoset ( Callithrix jacchus ) has attracted considerable attention, especially in the biomedical science and neuroscience research fields, because of its potential to recapitulate the complex and multidimensional phenotypes of human diseases, and several neurodegenerative transgenic models have been reported. However, there remain several issues as (i) it takes years to generate late-onset disease models, and (ii) the onset age and severity of phenotypes can vary among individuals due to differences in genetic background. In the present study, we established an efficient and rapid direct neuronal induction method (induced neurons; iNs) from embryonic and adult marmoset fibroblasts to investigate cellular-level phenotypes in the marmoset brain in vitro. We overexpressed reprogramming effectors, i.e., microRNA-9/9*, microRNA-124, and Achaete-Scute family bHLH transcription factor 1, in fibroblasts with a small molecule cocktail that facilitates neuronal induction. The resultant iNs from embryonic and adult marmoset fibroblasts showed neuronal characteristics within two weeks, including neuron-specific gene expression and spontaneous neuronal activity. As directly reprogrammed neurons have been shown to model neurodegenerative disorders, the neuronal reprogramming of marmoset fibroblasts may offer new tools for investigating neurological phenotypes associated with disease progression in non-human primate neurological disease models.

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.

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., (Copyright © 2019 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.)