Evolution of Public Opinion on COVID-19 Vaccination in Japan: Large-Scale Twitter Data Analysis.

BACKGROUND: Vaccines are promising tools to control the spread of COVID-19. An effective vaccination campaign requires government policies and community engagement, sharing experiences for social support, and voicing concerns about vaccine safety and efficiency. The increasing use of online social platforms allows us to trace large-scale communication and infer public opinion in real time. OBJECTIVE: This study aimed to identify the main themes in COVID-19 vaccine-related discussions on Twitter in Japan and track how the popularity of the tweeted themes evolved during the vaccination campaign. Furthermore, we aimed to understand the impact of critical social events on the popularity of the themes. METHODS: We collected more than 100 million vaccine-related tweets written in Japanese and posted by 8 million users (approximately 6.4% of the Japanese population) from January 1 to October 31, 2021. We used Latent Dirichlet Allocation to perform automated topic modeling of tweet text during the vaccination campaign. In addition, we performed an interrupted time series regression analysis to evaluate the impact of 4 critical social events on public opinion. RESULTS: We identified 15 topics grouped into the following 4 themes: (1) personal issue, (2) breaking news, (3) politics, and (4) conspiracy and humor. The evolution of the popularity of themes revealed a shift in public opinion, with initial sharing of attention over personal issues (individual aspect), collecting information from news (knowledge acquisition), and government criticism to focusing on personal issues. Our analysis showed that the Tokyo Olympic Games affected public opinion more than other critical events but not the course of vaccination. Public opinion about politics was significantly affected by various social events, positively shifting attention in the early stages of the vaccination campaign and negatively shifting attention later. CONCLUSIONS: This study showed a striking shift in public interest in Japan, with users splitting their attention over various themes early in the vaccination campaign and then focusing only on personal issues, as trust in vaccines and policies increased. An interrupted time series regression analysis showed that the vaccination rollout to the general population (under 65 years) increased the popularity of tweets about practical advice and personal vaccination experience, and the Tokyo Olympic Games disrupted public opinion but not the course of the vaccination campaign. The methodology developed here allowed us to monitor the evolution of public opinion and evaluate the impact of social events on public opinion, using large-scale Twitter data.

Functional Evaluation of Human Bioengineered Cardiac Tissue Using iPS Cells Derived from a Patient with Lamin Variant Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM) is caused by various gene variants and characterized by systolic dysfunction. Lamin variants have been reported to have a poor prognosis. Medical and device therapies are not sufficient to improve the prognosis of DCM with the lamin variants. Recently, induced pluripotent stem (iPS) cells have been used for research on genetic disorders. However, few studies have evaluated the contractile function of cardiac tissue with lamin variants. The aim of this study was to elucidate the function of cardiac cell sheet tissue derived from patients with lamin variant DCM. iPS cells were generated from a patient with lamin A/C (LMNA) -mutant DCM (LMNA p.R225X mutation). After cardiac differentiation and purification, cardiac cell sheets that were fabricated through cultivation on a temperature-responsive culture dish were transferred to the surface of the fibrin gel, and the contractile force was measured. The contractile force and maximum contraction velocity, but not the maximum relaxation velocity, were significantly decreased in cardiac cell sheet tissue with the lamin variant. A qRT-PCR analysis revealed that mRNA expression of some contractile proteins, cardiac transcription factors, Ca2+-handling genes, and ion channels were downregulated in cardiac tissue with the lamin variant.Human iPS-derived bioengineered cardiac tissue with the LMNA p.R225X mutation has the functional properties of systolic dysfunction and may be a promising tissue model for understanding the underlying mechanisms of DCM.

Glycan characteristics of human heart constituent cells maintaining organ function: relatively stable glycan profiles in cellular senescence.

Cell surface glycoproteins, which are good indicators of cellular types and biological function; are suited for cell evaluation. Tissue remodeling using various cells is a key feature of regenerative therapy. For artificial heart remodeling, a mixture of heart constituent cells has been investigated for organ assembly, however, the cellular characteristics remain unclear. In this study, the glycan profiles of human cardiomyocytes (HCMs), human cardiac fibroblasts (HCFs), and human vascular endothelial cells (ECs) were analyzed using evanescent-field lectin microarray analysis, a tool of glycan profiling, to clarify the required cellular characteristics. We found that ECs had more "α1-2fucose" and "core α1-6fucose" residues than other cells, and that "α2-6sialic acid" residue was more abundant in ECs and HCMs than in HCFs. HCFs showed higher abundance of "ß-galactose" and "ß-N-acetylgalactosamine" residues on N-glycan and O-glycan, respectively, compared to other cells. Interestingly, cardiac glycan profiles were insignificantly changed with cellular senescence. The residues identified in this study may participate in organ maintenance by contributing to the preservation of glycan components. Therefore, future studies should investigate the roles of glycans in optimal tissue remodeling since identifying cellular characteristics is important for the development of regenerative therapies.

Gangliosides as Signaling Regulators in Cancer.

At the plasma membrane, gangliosides, a group of glycosphingolipids, are expressed along with glycosphingolipids, phospholipids, and cholesterol in so-called lipid rafts that interact with signaling receptors and related molecules. Most cancers present abnormalities in the intracellular signal transduction system involved in tumor growth, invasion, and metastasis. To date, the roles of gangliosides as regulators of signal transduction have been reported in several cancer types. Gangliosides can be expressed by the exogenous ganglioside addition, with their endogenous expression regulated at the enzymatic level by targeting specific glycosyltransferases. Accordingly, the relationship between changes in the composition of cell surface gangliosides and signal transduction has been investigated by controlling ganglioside expression. In cancer cells, several types of signaling molecules are positively or negatively regulated by ganglioside expression levels, promoting malignant properties. Moreover, antibodies against gangliosides have been shown to possess cytotoxic effects on ganglioside-expressing cancer cells. In the present review, we highlight the involvement of gangliosides in the regulation of cancer cell signaling, and we explore possible therapies targeting ganglioside-expressing cancer.

Characterization of the metastatic potential of the floating cell component of MIA PaCa-2, a human pancreatic cancer cell line.

In pancreatic cancer, morphologically and functionally heterogeneous cancer cells reside within the same patient. The heterogeneity is believed to promote metastasis and resistance to chemoradiotherapy. MIA PaCa-2, an established human pancreatic ductal adenocarcinoma (PDAC) cell line, contains round and spindle-shaped adherent cells, as well as, round floating cells. In this study, we aimed to assess if the floating cells might have greater metastatic potential and/or be more resistant to drug-induced apoptosis compared to adherent cells. Time-lapse analysis revealed that the two types of adherent cells transformed bilaterally, and some of the adherent, round cells converted to floating cells. Flow cytometry and electron microscopy showed that approximately 90% of the floating cells were viable. qRT-PCR analysis revealed that floating cells expressed lower levels of integrins and ATP-binding cassette (ABC) transporters than adherent cells. In contrast, except for vimentin, floating cells expressed more epithelial to mesenchymal transition markers than adherent cells. Floating cells included a larger population of G2/M-phase cells, and migration assays revealed a decreased migration ability by floating cells relative to adherent cells. A cell aggregation assay showed that the aggregative properties of the floating cells were lower than those of the adherent cells. In 3D culture, spheres derived from floating cells were more sensitive to anti-cancer drugs, including gemcitabine, 5-FU, and abraxane, than those derived from adherent cells. Expression levels of stemness markers in the spheres derived from floating cells were lower than those derived from adherent cells. Morphological characterization of human PDAC cell lines may help to clarify the series of alterations cancer cells undergo during the metastatic process and may contribute to the development of new PDAC diagnostics and more patient-specific treatments for those with PDAC.

Mutations of histone demethylase genes encoded by X and Y chromosomes, Kdm5c and Kdm5d, lead to noncompaction cardiomyopathy in mice.

Mammalian X and Y chromosomes evolved from a pair of autosomes. Although most ancestral genes have been lost from the Y chromosome, a small number of ancestral X-Y gene pairs are still present on the sex chromosomes. The KDM5C and KDM5D genes, which encode H3K4 histone demethylases, are a surviving ancestral gene pair located on the X and Y chromosomes, respectively. Mutations in KDM5C cause X-linked intellectual disability in human males, suggesting functional divergence between KDM5C and KDM5D in the nervous system. In this study, to explore the functional conservation and divergence between these two genes in other organs, we generated female mice lacking Kdm5c (homozygous X5c- X5c- females) and male mice lacking both Kdm5c and Kdm5d (compound hemizygous X5c- Y5d- males). Both X5c- X5c- females and X5c- Y5d- males showed lower body weights and postnatal lethality. Histological examination of the hearts showed prominent trabecular extension and a thin layer of compacted myocardium in the left and right ventricles, indicating noncompaction cardiomyopathy. However, hemizygous males lacking either Kdm5c or Kdm5d showed no signs of noncompaction cardiomyopathy. These results clearly demonstrate that the function of Kdm5c and Kdm5d in heart development is conserved.

Rapamycin promotes endothelial-mesenchymal transition during stress-induced premature senescence through the activation of autophagy.

BACKGROUND: Rapamycin is known to be effective in suppressing senescence and the senescence-associated secretory phenotype (SASP). Therefore, it is highly expected to represent an anti-aging drug. Its anti-aging effect has been demonstrated at the mouse individual level. However, there are not many clinical findings with respect to its activity in humans. Here, we aimed to clarify the effect of rapamycin on human endothelial cells (ECs) as an in vitro model of human blood vessels. METHODS: Over the course of oxidative stress-induced senescence using hydrogen peroxide, we examined the effect of rapamycin on human coronary artery ECs (HCAECs). Senescence was evaluated by detecting senescence-associated ß-galactosidase (SA-ß-Gal) activity and the real-time PCR analysis of p16INK4a. Furthermore, expression levels of SASP factors were examined by real-time PCR and the expression of senescence-related antigens, such as intercellular adhesion molecule-1 (ICAM-1) and ganglioside GM1, were examined by fluorescence-activated cell sorting analysis and immunostaining. The inhibitory effect of rapamycin on mTOR signaling was examined by immunoblotting. The adhesion of leukocytes to HCAECs was evaluated by adhesion assays. Endothelial-mesenchymal transition (EndMT) induced by rapamycin treatment was evaluated by real-time PCR analysis and immunostaining for EndMT markers. Finally, we checked the activation of autophagy by immunoblotting and examined its contribution to EndMT by using a specific inhibitor. Furthermore, we examined how the activation of autophagy influences TGF-ß signaling by immunoblotting for Smad2/3 and Smad7. RESULTS: A decrease in SA-ß-Gal activity and the suppression of SASP factors were observed in HCAECs undergoing stress-induced premature senescence (SIPS) after rapamycin treatment. In contrast, ICAM-1 and ganglioside GM1 were upregulated by rapamycin treatment. In addition, leukocyte adhesion to HCAECs was promoted by this treatment. In rapamycin-treated HCAECs, morphological changes and the promotion of EndMT were also observed. Furthermore, we found that autophagy activation induced by rapamycin treatment, which led to activation of the TGF-ß pathway, contributed to EndMT induction. CONCLUSIONS: We revealed that although rapamycin functions to inhibit senescence and suppress SASP in HCAECs undergoing SIPS, EndMT is induced due to the activation of autophagy. Video abstract.

Application of Mesenchymal Stem Cell Therapy and Inner Ear Regeneration for Hearing Loss: A Review.

Inner and middle ear disorders are the leading cause of hearing loss, and are said to be among the greatest risk factors of dementia. The use of regenerative medicine for the treatment of inner ear disorders may offer a potential alternative to cochlear implants for hearing recovery. In this paper, we reviewed recent research and clinical applications in middle and inner ear regeneration and cell therapy. Recently, the mechanism of inner ear regeneration has gradually been elucidated. "Inner ear stem cells," which may be considered the precursors of various cells in the inner ear, have been discovered in the cochlea and vestibule. Research indicates that cells such as hair cells, neurons, and spiral ligaments may form promising targets for inner ear regenerative therapies by the transplantation of stem cells, including mesenchymal stem cells. In addition, it is necessary to develop tests for the clinical monitoring of cell transplantation. Real-time imaging techniques and hearing rehabilitation techniques are also being investigated, and cell therapy has found clinical application in cochlear implant techniques.

Fetal bovine serum enlarges the size of human pancreatic cancer spheres accompanied by an increase in the expression of cancer stem cell markers.

Pancreatic ductal adenocarcinoma (PDAC) is a major histological type of pancreatic cancer and remains one of the most lethal cancers with a high mortality rate owing to its aggressive growth, high metastatic rate, and recurrence. Recent studies on cancer stem cells (CSCs) have suggested that the aggressive growth, high metastatic rate, and recurrence might be caused by the ability of CSCs to self-renew, differentiate, and drive tumorigenesis. Thus, CSCs are expected to be a therapeutic target for PDAC. Sphere forming assay of cancer cells, including PDAC cells, is commonly performed using epidermal growth factor and fibroblast growth factor-2 containing serum-free medium to identify and isolate the enriched CSCs. Recently, we observed that PDAC spheres cultured in fetal bovine serum containing medium are morphologically similar to spheres cultured in the growth factor containing medium. In this study, we cultured two PDAC cell lines, PANC-1 and PK-1, in growth factor containing serum-free medium or serum containing medium, and compared the morphology of the spheres formed in detail by electron microscopy and examined the expression of major CSC marker genes. Both the PDAC cells formed larger spheres in the serum containing medium than in the growth factor containing medium. PK-1 cells formed more morphologically differentiated spheres, with peripheral flat lining cells, in the serum containing medium. Expression levels of most of the CSC markers were higher in the spheres of the two PDAC cells in both the culture mediums than in the cells cultured under adherent conditions. The expression levels of CSC markers in PDAC spheres cultured in the growth factor containing medium were not necessarily higher than that in the spheres cultured in the serum containing medium. These findings suggest that sphere forming assay using serum containing medium, by which large PDAC spheres with enriched CSCs are formed, may be useful for deciphering the characteristics of CSCs and for developing anti-CSC therapies for PDAC.

Vascular Diseases and Gangliosides.

Vascular diseases, such as myocardial infarction and cerebral infarction, are most commonly caused by atherosclerosis, one of the leading causes of death worldwide. Risk factors for atherosclerosis include lifestyle and aging. It has been reported that lifespan could be extended in mice by targeting senescent cells, which led to the suppression of aging-related diseases, such as vascular diseases. However, the molecular mechanisms underlying the contribution of aging to vascular diseases are still not well understood. Several types of cells, such as vascular (endothelial cell), vascular-associated (smooth muscle cell and fibroblast) and inflammatory cells, are involved in plaque formation, plaque rupture and thrombus formation, which result in atherosclerosis. Gangliosides, a group of glycosphingolipids, are expressed on the surface of vascular, vascular-associated and inflammatory cells, where they play functional roles. Clarifying the role of gangliosides in atherosclerosis and their relationship with aging is fundamental to develop novel prevention and treatment methods for vascular diseases based on targeting gangliosides. In this review, we highlight the involvement and possible contribution of gangliosides to vascular diseases and further discuss their relationship with aging.

Gangliosides Contribute to Vascular Insulin Resistance.

Insulin in physiological concentrations is important to maintain vascular function. Moreover, vascular insulin resistance contributes to vascular impairment. In the elderly, other factors including hypertension, dyslipidemia, and chronic inflammation amplify senescence of vascular endothelial and smooth muscle cells. In turn, senescence increases the risk for vascular-related diseases such as arteriosclerosis, diabetes, and Alzheimer's disease. Recently, it was found that GM1 ganglioside, one of the glycolipids localized on the cell membrane, mediates vascular insulin resistance by promoting senescence and/or inflammatory stimulation. First, it was shown that increased GM1 levels associated with aging/senescence contribute to insulin resistance in human aortic endothelial cells (HAECs). Second, the expression levels of gangliosides were monitored in HAECs treated with different concentrations of tumor necrosis factor-alpha (TNFα) for different time intervals to mimic in vivo acute or chronic inflammatory conditions. Third, the levels of insulin signaling-related molecules were monitored in HAECs after TNFα treatment with or without inhibitors of ganglioside synthesis. In this review, we summarize the molecular mechanisms of insulin resistance in aged/senescent and TNFα-stimulated endothelial cells mediated by gangliosides and highlight the possible roles of gangliosides in vascular insulin resistance-related diseases.

Stemness and anti-cancer drug resistance in ATP-binding cassette subfamily G member 2 highly expressed pancreatic cancer is induced in 3D culture conditions.

The expression of ATP-binding cassette subfamily G member 2 (ABCG2) is related to tumorigenic cancer stem cells (CSC) in several cancers. However, the effects of ABCG2 on CSC-related malignant characteristics in pancreatic ductal adenocarcinoma (PDAC) are not well elucidated. In this study, we compared the characteristics of low (ABCG2-) and high (ABCG2+)-ABCG2-expressing PDAC cells after cell sorting. In adherent culture condition, human PDAC cells, PANC-1, contained approximately 10% ABCG2+ cell populations, and ABCG2+ cells displayed more and longer microvilli compared with ABCG2- cells. Unexpectedly, ABCG2+ cells did not show significant drug resistance against fluorouracil, gemcitabine and vincristine, and ABCG2- cells exhibited higher sphere formation ability and stemness marker expression than those of ABCG2+ cells. Cell growth and motility was greater in ABCG2- cells compared with ABCG2+ cells. In contrast, epithelial-mesenchymal transition ability between ABCG2- and ABCG2+ cells was comparable. In 3D culture conditions, spheres derived from ABCG2- cells generated a large number of ABCG2+ cells, and the expression levels of stemness markers in these spheres were higher than spheres from ABCG2+ cells. Furthermore, spheres containing large populations of ABCG2+ cells exhibited high resistance against anti-cancer drugs presumably depending on ABCG2. ABCG2+ cells in PDAC in adherent culture are not correlated with stemness and malignant behaviors, but ABCG2+ cells derived from ABCG2- cells after sphere formation have stemness characteristics and anti-cancer drug resistance. These findings suggest that ABCG2- cells generate ABCG2+ cells and the malignant potential of ABCG2+ cells in PDAC varies depending on their environments.

Characteristic glycopeptides associated with extreme human longevity identified through plasma glycoproteomics.

BACKGROUND: Glycosylation is highly susceptible to changes of the physiological conditions, and accordingly, is a potential biomarker associated with several diseases and/or longevity. Semi-supercentenarians (SSCs; older than 105 years) are thought to be a model of human longevity. Thus, we performed glycoproteomics using plasma samples of SSCs, and identified proteins and conjugated N-glycans that are characteristic of extreme human longevity. METHODS: Plasma proteins from Japanese semi-supercentenarians (SSCs, 106-109 years), aged controls (70-88 years), and young controls (20-38 years) were analysed by using lectin microarrays and liquid chromatography/mass spectrometry (LC/MS). Peak area ratios of glycopeptides to corresponding normalising peptides were subjected to orthogonal projections to latent structures discriminant analysis (OPLS-DA). Furthermore, plasma levels of clinical biomarkers were measured. RESULTS: We found two lectins such as Phaseolus vulgaris, and Erythrina cristagalli (ECA), of which protein binding were characteristically increased in SSCs. Peak area ratios of ECA-enriched glycopeptides were successfully discriminated between SSCs and controls using OPLS-DA, and indicated that tri-antennary and sialylated N-glycans of haptoglobin at Asn207 and Asn211 sites were characterized in SSCs. Sialylated glycans of haptoglobin are a potential biomarker of several diseases, such as hepatocellular carcinoma, liver cirrhosis, and IgA-nephritis. However, the SSCs analysed here did not suffer from these diseases. CONCLUSIONS: Tri-antennary and sialylated N-glycans on haptoglobin at the Asn207 and Asn211 sites were abundant in SSCs and characteristic of extreme human longevity. GENERAL SIGNIFICANCE: We found abundant glycans in SSCs, which may be associated with human longevity.

Somatic mosaicism containing double mutations in PTCH1 revealed by generation of induced pluripotent stem cells from nevoid basal cell carcinoma syndrome.

BACKGROUND: Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterised by developmental defects and tumorigenesis, such as medulloblastomas and basal cell carcinomas, caused by mutations of the patched-1 (PTCH1) gene. In this article, we seek to demonstrate a mosaicism containing double mutations in PTCH1 in an individual with NBCCS. METHODS AND RESULTS: A de novo germline mutation of PTCH1 (c.272delG) was detected in a 31-year-old woman with NBCCS. Gene analysis of two out of four induced pluripotent stem cell (iPSC) clones established from the patient unexpectedly revealed an additional mutation, c.274delT. Deep sequencing confirmed a low-prevalence somatic mutation (5.5%-15.6% depending on the tissue) identical to the one found in iPSC clones. CONCLUSIONS: This is the first case of mosaicism unequivocally demonstrated in NBCCS. Furthermore, the mosaicism is unique in that the patient carries one normal and two mutant alleles. Because these mutations are located in close proximity, reversion error is likely to be involved in this event rather than a spontaneous mutation. In addition, this study indicates that gene analysis of iPSC clones can contribute to the detection of mosaicism containing a minor population carrying a second mutation.

A calcium-dependent protease as a potential therapeutic target for Wolfram syndrome.

Wolfram syndrome is a genetic disorder characterized by diabetes and neurodegeneration and considered as an endoplasmic reticulum (ER) disease. Despite the underlying importance of ER dysfunction in Wolfram syndrome and the identification of two causative genes, Wolfram syndrome 1 (WFS1) and Wolfram syndrome 2 (WFS2), a molecular mechanism linking the ER to death of neurons and ß cells has not been elucidated. Here we implicate calpain 2 in the mechanism of cell death in Wolfram syndrome. Calpain 2 is negatively regulated by WFS2, and elevated activation of calpain 2 by WFS2-knockdown correlates with cell death. Calpain activation is also induced by high cytosolic calcium mediated by the loss of function of WFS1. Calpain hyperactivation is observed in the WFS1 knockout mouse as well as in neural progenitor cells derived from induced pluripotent stem (iPS) cells of Wolfram syndrome patients. A small-scale small-molecule screen targeting ER calcium homeostasis reveals that dantrolene can prevent cell death in neural progenitor cells derived from Wolfram syndrome iPS cells. Our results demonstrate that calpain and the pathway leading its activation provides potential therapeutic targets for Wolfram syndrome and other ER diseases.

Ganglioside GM1 Contributes to the State of Insulin Resistance in Senescent Human Arterial Endothelial Cells.

Vascular endothelial cells (ECs) play central roles in physiologically important functions of blood vessels and contribute to the maintenance of vascular integrity. Therefore, it is considered that the impairment of EC functions leads to the development of vascular diseases. However, the molecular mechanisms of the EC dysfunctions that accompany senescence and aging have not yet been clarified. The carbohydrate antigens carried by glycoconjugates (e.g. glycoproteins, glycosphingolipids, and proteoglycans) mainly present on the cell surface serve not only as marker molecules but also as functional molecules. In this study, we have investigated the abundance and functional roles of glycosphingolipids in human ECs during senescence and aging. Among glycosphingolipids, ganglioside GM1 was highly expressed in abundance on the surface of replicatively and prematurely senescent ECs and also of ECs derived from an elderly subject. Insulin signaling, which regulates important functions of ECs, is impaired in senescent and aged ECs. Actually, by down-regulating GM1 on senescent ECs and overloading exogenous GM1 onto non-senescent ECs, we showed that an increased abundance of GM1 functionally contributes to the impairment of insulin signaling in ECs. Taken together, these findings provide the first evidence that GM1 increases in abundance on the cell surface of ECs under the conditions of cellular senescence and aging and causes insulin resistance in ECs. GM1 may be an attractive target for the detection, prevention, and therapy of insulin resistance and related vascular diseases, particularly in older people.

α2-6 sialylation is a marker of the differentiation potential of human mesenchymal stem cells.

Human somatic stem cells such as human mesenchymal stem cells (hMSCs) are considered attractive cell sources for stem cell-based therapy. However, quality control issues have been raised concerning their safety and efficacy. Here we used lectin microarray technology to identify cell surface glycans as markers of the differentiation potential of stem cells. We found that α2-6Sia-specific lectins show stronger binding to early passage adipose-derived hMSCs (with differentiation ability) than late passage cells (without the ability to differentiate). Flow cytometry analysis using α2-6Sia-specific lectins supported the results obtained by lectin microarray. Similar results were obtained for bone marrow-derived hMSCs and cartilage tissue-derived chondrocytes. Little or no binding of α2-6Sia-specific lectins was observed for human dermal fibroblasts, which are unable to differentiate, suggesting that the binding of α2-6Sia-specific lectins is associated with the differentiation ability of cells, but not to their capacity to proliferate. Quantitative analysis of the linkage mode of Sia using anion-exchange chromatography showed that the percentage of α2-6Sia linkage type was higher in early passage adipose-derived hMSCs than late passage cells. Integrinα5 was found to be a carrier protein of α2-6Sia. Sialidase treatment significantly reduced the differentiation efficiency of bone marrow-derived hMSCs. Based on these findings, we propose that α2-6sialylation is a marker of differentiation potential in stem cells such as adipose-derived hMSCs, bone marrow-derived hMSCs, and cartilage tissue-derived chondrocytes.

A synthetic nanofibrillar matrix promotes in vitro hepatic differentiation of embryonic stem cells and induced pluripotent stem cells.

Embryonic stem (ES) cells recapitulate normal developmental processes and serve as an attractive source for routine access to a large number of cells for research and therapies. We previously reported that ES cells cultured on M15 cells, or a synthesized basement membrane (sBM) substratum, efficiently differentiated into an endodermal fate and subsequently adopted fates of various digestive organs, such as the pancreas and liver. Here, we established a novel hepatic differentiation procedure using the synthetic nanofiber (sNF) as a cell culture scaffold. We first compared endoderm induction and hepatic differentiation between murine ES cells grown on sNF and several other substrata. The functional assays for hepatocytes reveal that the ES cells grown on sNF were directed into hepatic differentiation. To clarify the mechanisms for the promotion of ES cell differentiation in the sNF system, we focused on the function of Rac1, which is a Rho family member protein known to regulate the actin cytoskeleton. We observed the activation of Rac1 in undifferentiated and differentiated ES cells cultured on sNF plates, but not in those cultured on normal plastic plates. We also show that inhibition of Rac1 blocked the potentiating effects of sNF on endoderm and hepatic differentiation throughout the whole differentiation stages. Taken together, our results suggest that morphological changes result in cellular differentiation controlled by Rac1 activation, and that motility is not only the consequence, but is also able to trigger differentiation. In conclusion, we believe that sNF is a promising material that might contribute to tissue engineering and drug delivery.

Induction of primordial germ cell-like cells from mouse embryonic stem cells by ERK signal inhibition.

Primordial germ cells (PGCs) are embryonic germ cell precursors. Specification of PGCs occurs under the influence of mesodermal induction signaling during in vivo gastrulation. Although bone morphogenetic proteins and Wnt signaling play pivotal roles in both mesodermal and PGC specification, the signal regulating PGC specification remains unknown. Coculture of mouse embryonic stem cells (ESCs) with OP9 feeder cells induces mesodermal differentiation in vitro. Using this mesodermal differentiation system, we demonstrated that PGC-like cells were efficiently induced from mouse ESCs by extracellular signal-regulated kinase (ERK) signaling inhibition. Inhibition of ERK signaling by a MAPK/ERK kinase (MEK) inhibitor upregulated germ cell marker genes but downregulated mesodermal genes. In addition, the PGC-like cells showed downregulation of DNA methylation and formed pluripotent stem cell colonies upon treatment with retinoic acid. These results show that inhibition of ERK signaling suppresses mesodermal differentiation but activates germline differentiation program in this mesodermal differentiation system. Our findings provide a new insight into the signaling networks regulating PGC specification.

Pleiotropic functions of magnetic nanoparticles for ex vivo gene transfer.

Gene transfer technique has various applications, ranging from cellular biology to medical treatments for diseases. Although nonviral vectors, such as episomal vectors, have been developed, it is necessary to improve their gene transfer efficacy. Therefore, we attempted to develop a highly efficient gene delivery system combining an episomal vector with magnetic nanoparticles (MNPs). In comparison with the conventional method using transfection reagents, polyethylenimine-coated MNPs introduced episomal vectors more efficiently under a magnetic field and could express the gene in mammalian cells with higher efficiency and for longer periods. This novel in vitro separation method of gene-introduced cells utilizing the magnetic property of MNPs significantly facilitated the separation of cells of interest. Transplanted cells in vivo were detected using magnetic resonance. These results suggest that MNPs play multifunctional roles in ex vivo gene transfer, such as improvement of gene transfer efficacy, separation of cells, and detection of transplanted cells. FROM THE CLINICAL EDITOR: This study convincingly demonstrates enhanced efficiency of gene transfer via magnetic nanoparticles. The method also enables magnetic sorting of cells positive for the transferred gene, and in vivo monitoring of the process with MRI.