Generation of chimpanzee induced pluripotent stem cell lines for cross-species comparisons.

As humans' closest living relatives, chimpanzees offer valuable insights into human evolution. However, technical and ethical limitations hinder investigations into the molecular and cellular foundations that distinguish chimpanzee and human traits. Recently, induced pluripotent stem cells (iPSCs) have emerged as a novel model for functional comparative studies and provided a non-invasive alternative for studying embryonic phenomena. In this study, we generated five new chimpanzee iPSC lines from peripheral blood cells and skin fibroblasts with SeV vectors carrying four reprogramming factors (human OCT3/4, SOX2, KLF4, and L-MYC) and characterized their pluripotency and differentiation potential. We also examined the expression of a human-specific non-coding RNA, HSTR1, which is predicted to be involved in human brain development. Our results show that the chimpanzee iPSCs possess pluripotent characteristics and can differentiate into various cell lineages. Moreover, we found that HSTR1 is expressed in human iPSCs and their neural derivatives but not in chimpanzee counterparts, supporting its possible role in human-specific brain development. As iPSCs are inherently variable due to genetic and epigenetic differences in donor cells or reprogramming procedures, it is essential to expand the number of chimpanzee iPSC lines to comprehensively capture the molecular and cellular properties representative of chimpanzees. Hence, our cells provide a valuable resource for investigating the function and regulation of human-specific transcripts such as HSTR1 and for understanding human evolution more generally.

Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine.

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular mechanisms of disease, development, and evolution. To make primate induced pluripotent stem cells (iPSCs) more accessible, this paper presents a non-invasive method to generate human and non-human primate iPSCs from urine-derived cells, and their maintenance using a feeder-free culturing method. The urine can be sampled from a non-sterile environment (e.g., the cage of the animal) and treated with a broad-spectrum antibiotic cocktail during primary cell culture to reduce contamination efficiently. After propagation of the urine-derived cells, iPSCs are generated by a modified transduction method of a commercially available Sendai virus vector system. First iPSC colonies may already be visible after 5 days, and can be picked after 10 days at the earliest. Routine clump passaging with enzyme-free dissociation buffer supports pluripotency of the generated iPSCs for more than 50 passages.

Regulatory and coding sequences of TRNP1 co-evolve with brain size and cortical folding in mammals.

Brain size and cortical folding have increased and decreased recurrently during mammalian evolution. Identifying genetic elements whose sequence or functional properties co-evolve with these traits can provide unique information on evolutionary and developmental mechanisms. A good candidate for such a comparative approach is TRNP1, as it controls proliferation of neural progenitors in mice and ferrets. Here, we investigate the contribution of both regulatory and coding sequences of TRNP1 to brain size and cortical folding in over 30 mammals. We find that the rate of TRNP1 protein evolution (ω) significantly correlates with brain size, slightly less with cortical folding and much less with body size. This brain correlation is stronger than for >95% of random control proteins. This co-evolution is likely affecting TRNP1 activity, as we find that TRNP1 from species with larger brains and more cortical folding induce higher proliferation rates in neural stem cells. Furthermore, we compare the activity of putative cis-regulatory elements (CREs) of TRNP1 in a massively parallel reporter assay and identify one CRE that likely co-evolves with cortical folding in Old World monkeys and apes. Our analyses indicate that coding and regulatory changes that increased TRNP1 activity were positively selected either as a cause or a consequence of increases in brain size and cortical folding. They also provide an example how phylogenetic approaches can inform biological mechanisms, especially when combined with molecular phenotypes across several species.

Evaluation of occlusal status of Japanese adults based on functional tooth units.

OBJECTIVES: Although extensive national oral health data on dental caries and periodontal diseases in Japan are available, few studies have assessed the occlusal status of the Japanese population, and none are based on national survey data. The presence and prosthodontic conditions of the molar region are important for masticatory function, and the functional tooth unit (FTU) approach can be used to evaluate the occlusal status. Thus, using the national oral health survey data, this study investigated the occlusal status of the Japanese population using FTU. METHODS: Overall, 3,605 adults (aged ≥20 years) who participated in the 2011 Japanese national oral health survey were included. FTUs were used as indices for evaluating the occlusal status. FTUs were calculated according to sex, age group, and the number of teeth present, and their associations were further analysed. RESULTS: The number of teeth present, posterior teeth, and FTUs decreased with age in both men and women. In the age group of those ≥60 years, all only natural teeth-FTU (n-FTU) and natural teeth and artificial teeth from fixed prostheses or implant-supported FTU (nif-FTU) scores were <8. The total-FTU scores of all age groups, except the 60-69 and 70-79 years age groups, were >10. CONCLUSION: This is the first study to use FTUs and national oral health survey data to investigate the occlusal status in the Japanese population. People aged ≥60 years who have low n-FTU or natural teeth and artificial teeth from fixed prostheses or implant-supported FTU scores or those aged 60-70 years who have the lowest total-FTU scores require careful evaluation of masticatory performance.

The pluripotent stem cell-specific transcript ESRG is dispensable for human pluripotency.

Human pluripotent stem cells (PSCs) express human endogenous retrovirus type-H (HERV-H), which exists as more than a thousand copies on the human genome and frequently produces chimeric transcripts as long-non-coding RNAs (lncRNAs) fused with downstream neighbor genes. Previous studies showed that HERV-H expression is required for the maintenance of PSC identity, and aberrant HERV-H expression attenuates neural differentiation potentials, however, little is known about the actual of function of HERV-H. In this study, we focused on ESRG, which is known as a PSC-related HERV-H-driven lncRNA. The global transcriptome data of various tissues and cell lines and quantitative expression analysis of PSCs showed that ESRG expression is much higher than other HERV-Hs and tightly silenced after differentiation. However, the loss of function by the complete excision of the entire ESRG gene body using a CRISPR/Cas9 platform revealed that ESRG is dispensable for the maintenance of the primed and naïve pluripotent states. The loss of ESRG hardly affected the global gene expression of PSCs or the differentiation potential toward trilineage. Differentiated cells derived from ESRG-deficient PSCs retained the potential to be reprogrammed into induced PSCs (iPSCs) by the forced expression of OCT3/4, SOX2, and KLF4. In conclusion, ESRG is dispensable for the maintenance and recapturing of human pluripotency.

A non-invasive method to generate induced pluripotent stem cells from primate urine.

Comparing the molecular and cellular properties among primates is crucial to better understand human evolution and biology. However, it is difficult or ethically impossible to collect matched tissues from many primates, especially during development. An alternative is to model different cell types and their development using induced pluripotent stem cells (iPSCs). These can be generated from many tissue sources, but non-invasive sampling would decisively broaden the spectrum of non-human primates that can be investigated. Here, we report the generation of primate iPSCs from urine samples. We first validate and optimize the procedure using human urine samples and show that suspension- Sendai Virus transduction of reprogramming factors into urinary cells efficiently generates integration-free iPSCs, which maintain their pluripotency under feeder-free culture conditions. We demonstrate that this method is also applicable to gorilla and orangutan urinary cells isolated from a non-sterile zoo floor. We characterize the urinary cells, iPSCs and derived neural progenitor cells using karyotyping, immunohistochemistry, differentiation assays and RNA-sequencing. We show that the urine-derived human iPSCs are indistinguishable from well characterized PBMC-derived human iPSCs and that the gorilla and orangutan iPSCs are well comparable to the human iPSCs. In summary, this study introduces a novel and efficient approach to non-invasively generate iPSCs from primate urine. This will extend the zoo of species available for a comparative approach to molecular and cellular phenotypes.

Derivation of induced pluripotent stem cells in Japanese macaque (Macaca fuscata).

Non-human primates are our closest relatives and are of special interest for ecological, evolutionary and biomedical research. The Japanese macaque (Macaca fuscata) has contributed to the progress of primatology and neurosciences over 60 years. Despite this importance, the molecular and cellular basis of the Japanese macaque remains unexplored since useful cellular tools are lacking. Here we generated induced pluripotent stem cells (iPSCs) from skin fibroblasts of the Japanese macaque with Sendai virus or plasmid vectors. The Japanese macaque iPSCs (jm-iPSCs) were established under feeder-free culture conditions, but feeder cells turned out to be essential for their maintenance. The jm-iPSCs formed human iPSC-like flat colonies which were positive for pluripotent antigens including alkaline phosphatase, SSEA4, and TRA-1-81. They also expressed endogenous OCT3/4, SOX2, L-MYC, and KLF4 and other pluripotent marker genes. The potential to differentiate into all three germ layers and neural stem cells was confirmed by embryoid body and neurosphere formation, respectively. The jm-iPSCs will provide a robust in vitro tool for investigating the underlying mechanisms of development and physiology studies with the Japanese macaque.

Prevalence and risk factors of halitosis in Japanese school children.

BACKGROUND: Little data are available regarding halitosis in Japanese children. The aim of the current study was to investigate the prevalence and risk factors associated with halitosis in Japanese elementary and junior high school children. METHODS: The subjects consisted of 520 elementary (1st-6th grade: boys, n = 284; girls, n = 236) and 248 junior high (7th-9th grade: boys, n = 136; girls, n = 112) school children aged 6-15 years in Saitama Prefecture, Japan. A self-administered questionnaire survey; halitosis measurement using an organoleptic assessment method; and clinical oral examination were conducted. RESULTS: Overall, 44.9% of subjects had halitosis. The proportion of boys with halitosis was 43.6% and that of girls was 46.6%. On logistic regression analysis, grade and tongue coating were significant predictors of halitosis. The 7th-9th graders were significantly more likely to have halitosis than 1st-3rd graders (OR, 1.83; P = 0.007). Subjects with area of tongue coating score 2 or 3 were 5.51-fold more likely to present with halitosis (P < 0.001) than those with area of tongue coating score 0 or 1. Similarly, subjects with thickness of tongue coating score 2 or 3 were 3.28-fold more likely to have halitosis than those with thickness of tongue coating score 0 or 1 (P < 0.001). CONCLUSIONS: Halitosis in the school children is not a rare condition; instead, its occurrence is relatively high. Therefore, inclusion of a halitosis prevention and management component in school oral health programs would lead to the promotion of overall oral health.

Epigenetic Variation between Human Induced Pluripotent Stem Cell Lines Is an Indicator of Differentiation Capacity.

Variation in the differentiation capacity of induced pluripotent stem cells (iPSCs) to specific lineages is a significant concern for their use in clinical applications and disease modeling. To identify factors that affect differentiation capacity, we performed integration analyses between hematopoietic differentiation performance and molecular signatures such as gene expression, DNA methylation, and chromatin status, using 35 human iPSC lines and four ESC lines. Our analyses revealed that hematopoietic commitment of PSCs to hematopoietic precursors correlates with IGF2 expression level, which in turn depends on signaling-dependent chromatin accessibility at mesendodermal genes. Maturation capacity for conversion of PSC-derived hematopoietic precursors to mature blood associates with the amount and pattern of DNA methylation acquired during reprogramming. Our study therefore provides insight into the molecular features that determine the differential capacities seen among human iPSC lines and, through the predictive potential of this information, highlights a way to select optimal iPSCs for clinical applications.

Present and future challenges of induced pluripotent stem cells.

Growing old is our destiny. However, the mature differentiated cells making up our body can be rejuvenated to an embryo-like fate called pluripotency which is an ability to differentiate into all cell types by enforced expression of defined transcription factors. The discovery of this induced pluripotent stem cell (iPSC) technology has opened up unprecedented opportunities in regenerative medicine, disease modelling and drug discovery. In this review, we introduce the applications and future perspectives of human iPSCs and we also show how iPSC technology has evolved along the way.

Dynamic regulation of human endogenous retroviruses mediates factor-induced reprogramming and differentiation potential.

Pluripotency can be induced in somatic cells by overexpressing transcription factors, including POU class 5 homeobox 1 (OCT3/4), sex determining region Y-box 2 (SOX2), Krüppel-like factor 4 (KLF4), and myelocytomatosis oncogene (c-MYC). However, some induced pluripotent stem cells (iPSCs) exhibit defective differentiation and inappropriate maintenance of pluripotency features. Here we show that dynamic regulation of human endogenous retroviruses (HERVs) is important in the reprogramming process toward iPSCs, and in re-establishment of differentiation potential. During reprogramming, OCT3/4, SOX2, and KLF4 transiently hyperactivated LTR7s--the long-terminal repeats of HERV type-H (HERV-H)--to levels much higher than in embryonic stem cells by direct occupation of LTR7 sites genome-wide. Knocking down LTR7s or long intergenic non-protein coding RNA, regulator of reprogramming (lincRNA-RoR), a HERV-H-driven long noncoding RNA, early in reprogramming markedly reduced the efficiency of iPSC generation. KLF4 and LTR7 expression decreased to levels comparable with embryonic stem cells once reprogramming was complete, but failure to resuppress KLF4 and LTR7s resulted in defective differentiation. We also observed defective differentiation and LTR7 activation when iPSCs had forced expression of KLF4. However, when aberrantly expressed KLF4 or LTR7s were suppressed in defective iPSCs, normal differentiation was restored. Thus, a major mechanism by which OCT3/4, SOX2, and KLF4 promote human iPSC generation and reestablish potential for differentiation is by dynamically regulating HERV-H LTR7s.

Induction of pluripotency in human somatic cells via a transient state resembling primitive streak-like mesendoderm.

During mammalian embryonic development, the primitive streak initiates the differentiation of pluripotent epiblast cells into germ layers. Pluripotency can be reacquired in committed somatic cells using a combination of a handful of transcription factors, such as OCT3/4, SOX2, KLF4 and c-MYC (hereafter referred to as OSKM), albeit with low efficiency. Here we show that during OSKM-induced reprogramming towards pluripotency in human cells, intermediate cells transiently show gene expression profiles resembling mesendoderm, which is a major component of the primitive streak. Based on these findings, we discover that forkhead box H1 (FOXH1), a transcription factor required for anterior primitive streak specification during early development, significantly enhances the reprogramming efficiency of human fibroblasts by promoting their maturation, including mesenchymal to epithelial transition and the activation of late pluripotency markers. These results demonstrate that during the reprogramming process, human somatic cells go through a transient state that resembles mesendoderm.

Taste hyposensitivity in Japanese schoolchildren.

BACKGROUND: There is some research on taste disorder/hyposensitivity in special groups such as the elderly or patients presenting with specific taste problems, however few studies have been conducted among young populations. The objectives of this study were to estimate the prevalence of taste hyposensitivity and to investigate the relationship between taste hyposensitivity and oral health status in Japanese schoolchildren. METHODS: Subjects were 237 primary and 112 junior high school students in Saitama Prefecture, Japan. In total, 349 (boys: 181, girls: 168) students aged 6-15 years participated in the study. Oral examinations and whole-mouth taste tests using four tastes (sweet, salt, sour and bitter) solutions were conducted on the subjects. A subject who could not recognize the taste of the solution was defined as demonstrating hyposensitivity. RESULTS: Hyposensitivity was observed in 6.3% of all subjects for sweet-taste, 14.3% for salt-taste, 20.9% for sour-taste and 6.0% for bitter-taste. The prevalence of sweet, sour and bitter-taste hyposensitivity decreased as the subjects' grade advanced. In contrast, the prevalence of salt-taste hyposensitivity increased in 7th-9th grade subjects. Furthermore, the prevalence of bitter-taste hyposensitivity was significantly higher in males than females among 1st-3rd graders.Taste hyposensitivity had little association with oral health status, such as decayed teeth, filled teeth, dental plaque, gingival status and tongue coating. CONCLUSIONS: In this study, taste hyposensitivity was observed in 6.0%-20.9% of the students. There was little association between taste hyposensitivity and oral health status. The current study implies that the factors affecting the taste hyposensitivity in children may different from those in the elderly. Therefore it is necessary to further investigate the causes of taste hyposensitivity among younger generation.

Differentiation-defective phenotypes revealed by large-scale analyses of human pluripotent stem cells.

We examined the gene expression and DNA methylation of 49 human induced pluripotent stem cells (hiPSCs) and 10 human embryonic stem cells and found overlapped variations in gene expression and DNA methylation in the two types of human pluripotent stem cell lines. Comparisons of the in vitro neural differentiation of 40 hiPSCs and 10 human embryonic stem cells showed that seven hiPSC clones retained a significant number of undifferentiated cells even after neural differentiation culture and formed teratoma when transplanted into mouse brains. These differentiation-defective hiPSC clones were marked by higher expression levels of several genes, including those expressed from long terminal repeats of specific human endogenous retroviruses. These data demonstrated a subset of hiPSC lines that have aberrant gene expression and defective potential in neural differentiation, which need to be identified and eliminated before applications in regenerative medicine.

Functional tooth units and nutritional status of older people in care homes in Indonesia.

OBJECTIVES: To investigate the relationship between functional tooth units (FTUs) and nutritional status. METHODS: One hundred females (mean age: 72.4 ± 8.2 years) at four private care homes in Jakarta, Indonesia were interviewed and clinically examined. The oral examination included the assessment of teeth, prosthetic status, and number of FTUs. The total number of FTUs was further divided by tooth composition: natural tooth against natural tooth (NN-FTUs), natural tooth against denture (ND-FTUs), and denture against denture (DD-FTUs). Nutritional status was evaluated using the body mass index (BMI) and the Mini Nutritional Assessment (MNA). RESULTS: The mean numbers of teeth present, NN-FTUs, ND-FTUs, DD-FTUs, and total FTUs were 13.1 ± 10.4, 1.7 ± 3.0, 1.2 ± 3.3, 0.4 ± 1.2 and 3.3 ± 4.4, respectively. The mean BMI and MNA scores were 24.8 ± 5.0 and 22.6 ± 2.8, respectively. Subjects with a normal BMI had a significantly higher total number of FTUs (3.6 ± 4.6) compared with underweight subjects (0.1 ± 0.3). Subjects with a normal MNA had a significantly higher number of NN-FTU (2.6 ± 3.7) compared to those who were at risk or in a state of under-nutrition (1.2 ± 2.4). CONCLUSION: This study revealed significant relationships between the number of FTUs and nutritional status. Keeping the posterior occlusion should be emphasized in order to maintain good nutritional status in older subjects.

Exploring taste hyposensitivity in Japanese senior high school students.

AIM: The main objective of this study was to investigate the prevalence of taste hyposensitivity and the relationships between sex, oral health status, and eating habits with taste hyposensitivity in Japanese senior high school students. METHODS: Oral examinations, sweet and salt whole-mouth taste tests, and a questionnaire about eating habits were conducted on 234 senior high school students. Factors affecting taste hyposensitivity were investigated using a multivariate analysis. RESULTS: Sweet-taste hyposensitivity was observed in 7.3% of the students, and salt-taste hyposensitivity in 22.2%. Approximately 3% of the students had both sweet- and salt-taste hyposensitivity, and 22.6% had either sweet- or salt-taste hyposensitivity. In total, 26% had a taste hyposensitivity. There were significant relationships between the intake of instant noodles with sweet-taste hyposensitivity, and the intake of vegetables or isotonic drinks with salt-taste hyposensitivity. CONCLUSIONS: There was a significant association between eating habits and taste hyposensitivity in Japanese senior high school students. Taste tests would be a helpful adjunct for students to recognize variations in taste sensitivity, and a questionnaire about their eating habits might provide an effective self-review of their eating habits, and therefore, provide motivation to change.

Oral malodor and related factors in Japanese senior high school students.

BACKGROUND: Oral malodor (halitosis or bad breath) might be an important motivation tool for improving oral health in adolescents. There are few studies that report the epidemiology of oral malodor in high school students and the relationships with lifestyle and oral health status. This research was conducted to obtain underlying data for introducing an oral health education program which targeted prevention of oral malodor as a motivation tool for changing oral health behavior in high school students. METHODS: A questionnaire, school oral examination, and oral malodor measurement were conducted on senior high school students in a Tokyo metropolitan school in 2007. A total of 474 students (male: 219, female: 255) were used for the analysis. RESULTS: Over 42% of subjects reported that they had experienced anxiety, or were conscious of oral malodor, on at least 1 occasion. The students who had detectable oral malodor comprised 39.6% of subjects. The binary logistic regression analyses showed that whether or not subjects ate breakfast before the oral examination (p < .05), the presence of plaque (p < .01), and presence of a substantive tongue coating (p < .01) were related to the presence of detectable oral malodor. CONCLUSIONS: Cleaning the oral cavity and eating breakfast are important to prevent oral malodor in high school students. This study indicated that school health education incorporating prevention of oral malodor as a motivation tool for oral health promotion could be a valuable procedure to include in high school dental health education programs.

Effects of a mouthwash with chlorine dioxide on oral malodor and salivary bacteria: a randomized placebo-controlled 7-day trial.

BACKGROUND: Previous research has shown the oxidizing properties and microbiological efficacies of chlorine dioxide (ClO2). Its clinical efficacies on oral malodor have been evaluated and reported only in short duration trials, moreover, no clinical studies have investigated its microbiological efficacies on periodontal and malodorous bacteria. Thus, the aim of this study was to assess the inhibitory effects of a mouthwash containing ClO2 used for 7 days on morning oral malodor and on salivary periodontal and malodorous bacteria. METHODS/DESIGN: A randomized, double blind, crossover, placebo-controlled trial was conducted among 15 healthy male volunteers, who were divided into 2 groups. Subjects were instructed to rinse with the experimental mouthwash containing ClO2 or the placebo mouthwash, without ClO2, twice per day for 7 days. After a one week washout period, each group then used the opposite mouthwash for 7 days. At baseline and after 7 days, oral malodor was evaluated with Organoleptic measurement (OM), and analyzed the concentrations of hydrogen sulfide (H2S), methyl mercaptan (CH3SH) and dimethyl sulfide ((CH3)2S), the main VSCs of human oral malodor, were assessed by gas chromatography (GC). Clinical outcome variables included plaque and gingival indices, and tongue coating index. The samples of saliva were microbiologically investigated. Quantitative and qualitative analyses were performed using the polymerase chain reaction-Invader method. RESULTS AND DISCUSSION: The baseline oral condition in healthy subjects in the 2 groups did not differ significantly. After rinsing with the mouthwash containing ClO2 for 7 days, morning bad breath decreased as measured by the OM and reduced the concentrations of H2S, CH3SH and (CH3)2S measured by GC, were found. Moreover ClO2 mouthwash used over a 7-day period appeared effective in reducing plaque, tongue coating accumulation and the counts of Fusobacterium nucleatum in saliva. Future research is needed to examine long-term effects, as well as effects on periodontal diseases and plaque accumulation in a well-defined sample of halitosis patients and broader population samples. TRIAL REGISTRATION: ClinicalTrials.gov NCT00748943.

Variation in the safety of induced pluripotent stem cell lines.

We evaluated the teratoma-forming propensity of secondary neurospheres (SNS) generated from 36 mouse induced pluripotent stem (iPS) cell lines derived in 11 different ways. Teratoma-formation of SNS from embryonic fibroblast-derived iPS cells was similar to that of SNS from embryonic stem (ES) cells. In contrast, SNS from iPS cells derived from different adult tissues varied substantially in their teratoma-forming propensity, which correlated with the persistence of undifferentiated cells.

Generation and characterization of human induced pluripotent stem cells.

This unit describes how to generate human induced pluripotent stem (iPS) cells and evaluate the qualities of the generated iPS cells. The methods for establishment and maintenance of human iPS cells are similar to those for mouse iPS cells but not identical. In addition, these protocols include excellent procedures for passaging and cryopreservation of human iPS cells established by ES cell researchers, which result in an easy way to culture human iPS cells. Moreover, we include methods for characterizing iPS cells for further research. RT-PCR and immunocytochemistry for detection of pluripotent cell markers, embryoid body differentiation, and teratoma differentiation are used to determine pluripotency in vitro and in vivo, respectively.