Follicular helper T (Tfh) cells within secondary lymphoid organs control multiple steps of B cell maturation and antibody (Ab) production. HIV-1 infection is associated with an altered B cell differentiation and Tfh isolated from lymph nodes of HIV-infected (HIV+) individuals provide inadequate B cell help in vitro. However, the mechanisms underlying this impairment of Tfh function are not fully defined. Using a unique collection of splenocytes, we compared the frequency, phenotype and transcriptome of Tfh subsets in spleens from HIV negative (HIV-) and HIV+ subjects. We observed an increase of CXCR5+PD-1highCD57-Tfh and germinal center (GC) CD57+ Tfh in HIV+ spleens. Both subsets showed a reduced mRNA expression of the transcription factor STAT-3, co-stimulatory, regulatory and signal transduction molecules as compared to HIV- spleens. Similarly, Foxp3 expressing follicular regulatory T (Tfr) cells were increased, suggesting sustained GC reactions in chronically HIV+ spleens. As a consequence, GC B cell populations were expanded, however, complete maturation into memory B cells was reduced in HIV+ spleens where we evidenced a compromised production of B cell-activating cytokines such as IL-4 and IL-10. Collectively our data indicate that, although Tfh proliferation and GC reactions seem to be ongoing in HIV-infected spleens, Tfh "differentiation" and expression of costimulatory molecules is skewed with a profound effect on B cell maturation.

Recent evidence suggest that a β-glucan derived from mushroom Schizophyllan(SPG) complexed with a humanized TLR9 agonistic CpG DNA, K3 (K3-SPG) is a promising vaccine adjuvant that induces robust CD8 T cell responses to co-administered antigen. However, it has not been investigated whether K3-SPG alone can act as an anti-cancer immunotherapeutic agent or not. Here, we demonstrate that intravenous injection of K3-SPG, but not CpG alone, is accumulated in the tumor microenvironment and triggered immunogenic cell death (ICD) of tumor cells by local induction of type-I interferon (IFN) as well as IL-12. Resultant innate immune activation as well as subsequent tumor-specific CD8 T cell responses were contributed the tumor growth suppression. This anti-tumor effect of K3-SPG monotherapy was also confirmed by using various tumor models including pancreatic cancer peritoneal dissemination model. Taken together, nano-particulate TLR9 agonist injected intravenously can scout out tumor microenvironment to provoke local innate immune activation and release dead tumor cells into circulation that may induce broader and protective tumor antigen-specific CD8 T cells.

Rapid and dependable methods for isolating human pluripotent stem cell (hPSC) populations are urgently needed for quality control in basic research and in cell-based therapy applications. Using lectin arrays, we analyzed glycoproteins extracted from 26 hPSC samples and 22 differentiated cell samples, and identified a small group of lectins with distinctive binding signatures that were sufficient to distinguish hPSCs from a variety of non-pluripotent cell types. These specific biomarkers were shared by all the 12 human embryonic stem cell and the 14 human induced pluripotent stem cell samples examined, regardless of the laboratory of origin, the culture conditions, the somatic cell type reprogrammed, or the reprogramming method used. We demonstrated a practical application of specific lectin binding by detecting hPSCs within a differentiated cell population with lectin-mediated staining followed by fluorescence microscopy and flow cytometry, and by enriching and purging viable hPSCs from mixed cell populations using lectin-mediated cell separation. Global gene expression analysis showed pluripotency-associated differential expression of specific fucosyltransferases and sialyltransferases, which may underlie these differences in protein glycosylation and lectin binding. Taken together, our results show that protein glycosylation differs considerably between pluripotent and non-pluripotent cells, and demonstrate that lectins may be used as biomarkers to monitor pluripotency in stem cell populations and for removal of viable hPSCs from mixed cell populations.

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.

In order to apply human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to regenerative medicine, the cells should be produced under restricted conditions conforming to GMP guidelines. Since the conventional culture system has some issues that need to be addressed to achieve this goal, we developed a novel culture system. We found that recombinant laminin-511 E8 fragments are useful matrices for maintaining hESCs and hiPSCs when used in combination with a completely xeno-free (Xf) medium, StemFit™. Using this system, hESCs and hiPSCs can be easily and stably passaged by dissociating the cells into single cells for long periods, without any karyotype abnormalities. Human iPSCs could be generated under feeder-free (Ff) and Xf culture systems from human primary fibroblasts and blood cells, and they possessed differentiation abilities. These results indicate that hiPSCs can be generated and maintained under this novel Ff and Xf culture system.

Experimental animal models have played an indispensable role in the development of human induced pluripotent stem cell (iPSC) research. The derivation of high-quality (so-called "true naïve state") iPSCs of non-human primates enhances their application and safety for human regenerative medicine. Although several attempts have been made to convert human and non-human primate PSCs into a truly naïve state, it is unclear which evaluation methods can discriminate them as being truly naïve. Here we attempted to derive naïve cynomolgus monkey (Cm) (Macaca fascicularis) embryonic stem cells (ESCs) and iPSCs. Several characteristics of naïve Cm ESCs including colony morphology, appearance of naïve-related mRNAs and proteins, leukaemia inhibitory factor dependency, and mitochondrial respiration were confirmed. Next, we generated Cm iPSCs and converted them to a naïve state. Transcriptomic comparison of PSCs with early Cm embryos elucidated the partial achievement (termed naïve-like) of their conversion. When these were subjected to in vitro neural differentiation, enhanced differentiating capacities were observed after naïve-like conversion, but some lines exhibited heterogeneity. The difficulty of achieving contribution to chimeric mouse embryos was also demonstrated. These results suggest that Cm PSCs could ameliorate their in vitro neural differentiation potential even though they could not display true naïve characteristics.

Spinocerebellar ataxia type 36 is a late-onset, slowly progressive cerebellar syndrome with motor neuron degeneration that is caused by expansions of a hexanucleotide repeat (GGCCTG) in the noncoding region of NOP56 gene, with a histopathological feature of RNA foci formation in postmortem tissues. Here, we report a cellular model using the spinocerebellar ataxia type 36 patient induced pluripotent stem cells (iPSCs). We generated iPSCs from spinocerebellar ataxia type 36 patients and differentiated them into neurons. The number of RNA-foci-positive cells was increased in patient iPSCs and iPSC-derived neurons. Treatment of the 2'-O, 4'-C-ethylene-bridged nucleic acid antisense oligonucleotides (ASOs) targeting NOP56 pre-mRNA reduced RNA-foci-positive cells to ∼50% in patient iPSCs and iPSC-derived neurons. NOP56 mRNA expression levels were lower in patient iPSCs and iPSC-derived neurons than in healthy control neurons. One of the ASOs reduced the number of RNA-foci-positive cells without altering NOP56 mRNA expression levels in patient iPSCs and iPSC-derived neurons. These data show that iPSCs from spinocerebellar ataxia type 36 patients can be useful for evaluating the effects of ASOs toward GGCCTG repeat expansion in spinocerebellar ataxia type 36.

Despite the growing evidence for the regulated spindle orientation in mammals, a systematic approach for identifying the responsible genes in mammalian cells has not been established. Here we perform a kinase-targeting RNAi screen in HeLa cells and identify ABL1 as a novel regulator of spindle orientation. Knockdown of ABL1 causes the cortical accumulation of Leu-Gly-Asn repeat-enriched-protein (LGN), an evolutionarily conserved regulator of spindle orientation. This results in the LGN-dependent spindle rotation and spindle misorientation. In vivo inactivation of ABL1 by a pharmacological inhibitor or by ablation of the abl1 gene causes spindle misorientation and LGN mislocalization in mouse epidermis. Furthermore, ABL1 directly phosphorylates NuMA, a binding partner of LGN, on tyrosine 1774. This phosphorylation maintains the cortical localization of NuMA during metaphase, and ensures the LGN/NuMA-dependent spindle orientation control. This study provides a novel approach to identify genes regulating spindle orientation in mammals and uncovers new signalling pathways for this mechanism.

Intermittent fasting is one of the most effective dietary restriction regimens that extend life span in C. elegans and mammals. Fasting-stimulus responses are key to the longevity response; however, the mechanisms that sense and transduce the fasting stimulus remain largely unknown. Through a comprehensive transcriptome analysis in C. elegans, we find that along with the FOXO transcription factor DAF-16, AP-1 (JUN-1/FOS-1) plays a central role in fasting-induced transcriptional changes. KGB-1, one of the C. elegans JNKs, acts as an activator of AP-1 and is activated in response to fasting. KGB-1 and AP-1 are involved in intermittent fasting-induced longevity. Fasting-induced upregulation of the components of the SCF E3 ubiquitin ligase complex via AP-1 and DAF-16 enhances protein ubiquitination and reduces protein carbonylation. Our results thus identify a fasting-responsive KGB-1/AP-1 signaling pathway, which, together with DAF-16, causes transcriptional changes that mediate longevity, partly through regulating proteostasis.

Induction of a series of anti-hypoxic proteins protects cells during exposure to hypoxic conditions. Hypoxia-inducible factor-α (HIF-α) is a major transcription factor that orchestrates this protective effect. To activate HIF exogenously, without exposing cells to hypoxic conditions, many small-molecule inhibitors targeting prolyl hydroxylase domain-containing protein have been developed. In addition, suppression of factor inhibiting HIF-1 (FIH-1) has also been shown to have the potential to activate HIF-α. However, few small-molecule inhibitors of FIH-1 have been developed. In this study, we synthesized a series of furan- and thiophene-2-carbonyl amino acid derivatives having the potential to inhibit FIH-1. The inhibitory activities of these compounds were evaluated in SK-N-BE(2)c cells by measuring HIF response element (HRE) promoter activity. Several furan- and thiophene-2-carbonyl amino acid derivatives inhibited FIH-1 based on correlations among the docking score of the FIH-1 active site, the chemical structure of the compounds, and biological HIF-α/HRE transcriptional activity.

Allograft bone is a widely used as a convenient tool for reconstructing massive bone defects in orthopedic surgery. However, allografts are associated with the risk of viral disease transmission. One of the viruses transmitted in this manner is human T-lymphotropic virus type 1 (HTLV-1), which is found worldwide but is unevenly distributed. The southwestern parts of Japan are a highly endemic for HTLV-1. We investigated the HTLV-1 seroprevalence in candidate allograft donors at the regional bone bank in Kagoshima, Japan during its first 5 years of service. Between 2008 and 2012, we collected 282 femoral heads at the Kagoshima regional bone bank from living donors with osteoarthritis of the hip joint. Among the 282 candidate donors, 32 donors (11.3 %) were seropositive for anti-HTLV-1 antibody; notably, this prevalence is higher than that reported for blood donors in this area. Additionally, to determine if HTLV-1 genes are detectable after processing, we examined the bone marrow of the femoral heads from seropositive donors by conducting PCR assays. Our results confirm the existence of viral genes following the heat treatment processing of the femoral heads. Therefore, it is important to inactivate a virus completely by heat-treatment. Together, our findings highlight the importance of HTLV-1 screening at bone banks, particularly in HTLV-1-endemic areas such as southwest Japan.

The blood-brain barrier (BBB) is composed of four cell populations, brain endothelial cells (BECs), pericytes, neurons, and astrocytes. Its role is to precisely regulate the microenvironment of the brain through selective substance crossing. Here we generated an in vitro model of the BBB by differentiating human induced pluripotent stem cells (hiPSCs) into all four populations. When the four hiPSC-derived populations were co-cultured, endothelial cells (ECs) were endowed with features consistent with BECs, including a high expression of nutrient transporters (CAT3, MFSD2A) and efflux transporters (ABCA1, BCRP, PGP, MRP5), and strong barrier function based on tight junctions. Neuron-derived Dll1, which activates Notch signaling in ECs, was essential for the BEC specification. We performed in vitro BBB permeability tests and assessed ten clinical drugs by nanoLC-MS/MS, finding a good correlation with the BBB permeability reported in previous cases. This technology should be useful for research on human BBB physiology, pathology, and drug development.

De novo establishment of DNA methylation is accomplished by DNMT3A and DNMT3B. Here, we analyze de novo DNA methylation in mouse embryonic fibroblasts (2i-MEFs) derived from DNA-hypomethylated 2i/L ES cells with genetic ablation of Dnmt3a or Dnmt3b. We identify 355 and 333 uniquely unmethylated genes in Dnmt3a and Dnmt3b knockout (KO) 2i-MEFs, respectively. We find that Dnmt3a is exclusively required for de novo methylation at both TSS regions and gene bodies of Polycomb group (PcG) target developmental genes, while Dnmt3b has a dominant role on the X chromosome. Consistent with this, tissue-specific DNA methylation at PcG target genes is substantially reduced in Dnmt3a KO embryos. Finally, we find that human patients with DNMT3 mutations exhibit reduced DNA methylation at regions that are hypomethylated in Dnmt3 KO 2i-MEFs. In conclusion, here we report a set of unique de novo DNA methylation target sites for both DNMT3 enzymes during mammalian development that overlap with hypomethylated sites in human patients.

CpG islands (CGIs) including those at imprinting control regions (ICRs) are protected from de novo methylation in somatic cells. However, many cancers often exhibit CGI hypermethylation, implying that the machinery is impaired in cancer cells. Here, we conducted a comprehensive analysis of CGI methylation during somatic cell reprogramming. Although most CGIs remain hypomethylated, a small subset of CGIs, particularly at several ICRs, was often de novo methylated in reprogrammed pluripotent stem cells (PSCs). Such de novo ICR methylation was linked with the silencing of reprogramming factors, which occurs at a late stage of reprogramming. The ICR-preferred CGI hypermethylation was similarly observed in human PSCs. Mechanistically, ablation of Dnmt3a prevented PSCs from de novo ICR methylation. Notably, the ICR-preferred CGI hypermethylation was observed in pediatric cancers, while adult cancers exhibit genome-wide CGI hypermethylation. These results may have important implications in the pathogenesis of pediatric cancers and the application of PSCs.

Endothelial cells (ECs) line blood vessels and serve as a niche for hematopoietic stem and progenitor cells (HSPCs). Recent data point to tissue-specific EC specialization as well as heterogeneity; however, it remains unclear how ECs acquire these properties. Here, by combining live-imaging-based lineage-tracing and single-cell transcriptomics in zebrafish embryos, we identify an unexpected origin for part of the vascular HSPC niche. We find that islet1 (isl1)-expressing cells are the progenitors of the venous ECs that constitute the majority of the HSPC niche. These isl1-expressing cells surprisingly originate from the endoderm and differentiate into ECs in a process dependent on Bmp-Smad signaling and subsequently requiring npas4l (cloche) function. Single-cell RNA sequencing analyses show that isl1-derived ECs express a set of genes that reflect their distinct origin. This study demonstrates that endothelial specialization in the HSPC niche is determined at least in part by the origin of the ECs.

Understanding the T-cell responses involved in inhibiting COVID-19 severity is crucial for developing new therapeutic and vaccine strategies. Here, we characterized SARS-CoV-2 spike-specific CD8+ T cells in vaccinees longitudinally. The BNT162b2 mRNA vaccine can induce spike-specific CD8+ T cells cross-reacting to BA.1, whereas the T-cell receptor (TCR) repertoire usages decreased with time. Furthermore the mRNA vaccine induced spike-specific CD8+ T cells subpopulation expressing Granzyme A (GZMA), Granzyme B (GZMB) and Perforin simultaneously in healthy donors at 4 weeks after the second vaccination. The induced subpopulation was not maintained at 12 weeks after the second vaccination. Incorporating factors that efficiently induce CD8+ T cells with highly cytotoxic activity could improve future vaccine efficacy against such variants.

The isolation and characterization of neutralizing antibodies from infection and vaccine settings informs future vaccine design, and methodologies that streamline the isolation of antibodies and the generation of B cell clones are of great interest. Retroviral transduction to express Bcl-6 and Bcl-xL and transform primary B cells has been shown to promote long-term B cell survival and antibody secretion in vitro, and can be used to isolate antibodies from memory B cells. However, application of this methodology to B cell subsets from different tissues and B cells from chronically infected individuals has not been well characterized. Here, we characterize Bcl-6/Bcl-xL B cell immortalization across multiple tissue types and B cell subsets in healthy and HIV-1 infected individuals, as well as individuals recovering from malaria. In healthy individuals, naïve and memory B cell subsets from PBMCs and tonsil tissue transformed with similar efficiencies, and displayed similar characteristics with respect to their longevity and immunoglobulin secretion. In HIV-1-viremic individuals or in individuals with recent malaria infections, the exhausted CD27-CD21- memory B cells transformed with lower efficiency, but the transformed B cells expanded and secreted IgG with similar efficiency. Importantly, we show that this methodology can be used to isolate broadly neutralizing antibodies from HIV-infected individuals. Overall, we demonstrate that Bcl-6/Bcl-xL B cell immortalization can be used to isolate antibodies and generate B cell clones from different B cell populations, albeit with varying efficiencies.

Reactive oxygen species (ROS) produced by NADPH1 oxidase 1 (NOX1) are thought to drive spermatogonial stem cell (SSC) self-renewal through feed-forward production of ROS by the ROS-BCL6B-NOX1 pathway. Here we report the critical role of oxygen on ROS-induced self-renewal. Cultured SSCs proliferated poorly and lacked BCL6B expression under hypoxia despite increase in mitochondria-derived ROS. Due to lack of ROS amplification under hypoxia, NOX1-derived ROS were significantly reduced, and Nox1-deficient SSCs proliferated poorly under hypoxia but normally under normoxia. NOX1-derived ROS also influenced hypoxic response in vivo because Nox1-deficient undifferentiated spermatogonia showed significantly reduced expression of HIF1A, a master transcription factor for hypoxic response. Hypoxia-induced poor proliferation occurred despite activation of MYC and suppression of CDKN1A by HIF1A, whose deficiency exacerbated self-renewal efficiency. Impaired proliferation of Nox1- or Hif1a-deficient SSCs under hypoxia was rescued by Cdkn1a depletion. Consistent with these observations, Cdkn1a-deficient SSCs proliferated actively only under hypoxia but not under normoxia. On the other hand, chemical suppression of mitochondria-derived ROS or Top1mt mitochondria-specific topoisomerase deficiency did not influence SSC fate, suggesting that NOX1-derived ROS play a more important role in SSCs than mitochondria-derived ROS. These results underscore the importance of ROS origin and oxygen tension on SSC self-renewal.

Nano-sized metal particles are attracting much interest in industrial and biomedical applications due to the recent progress and development of nanotechnology, and the surface-modifications by appropriate polymers are key techniques to stably express their characteristics. Herein, we applied cyclic poly(ethylene glycol) (c-PEG), having no chemical inhomogeneity, to provide a polymer topology-dependent stabilization for the surface-modification of gold nanoparticles (AuNPs) through physisorption. By simply mixing c-PEG, but not linear counterparts, enables AuNPs to maintain dispersibility through freezing, lyophilization, or heating. Surprisingly, c-PEG endowed AuNPs with even better dispersion stability than thiolated PEG (HS-PEG-OMe). The stronger affinity of c-PEG was confirmed by DLS, ζ-potential, and FT-IR. Furthermore, the c-PEG system exhibited prolonged blood circulation and enhanced tumor accumulation in mice. Our data suggests that c-PEG induces physisorption on AuNPs, supplying sufficient stability toward bio-medical applications, and would be an alternative approach to the gold-sulfur chemisorption.

ES cell (ESC) identity is stably maintained through the coordinated regulation of transcription factors and chromatin structure. SMARCB1, also known as INI1, SNF5, BAF47, is one of the subunits of SWI/SNF (BAF) complexes that play a crucial role in regulating gene expression by controlling chromatin dynamics. Genetic ablation of Smarcb1 in mice leads to embryonic lethality at the peri-implantation stage, indicating that Smarcb1 is important for the early developmental stages. However, the role of SMARCB1 in the maintenance of the ESC identity remains unknown. Here we established mouse ESCs lacking Smarcb1 and investigated the effect of Smarcb1 ablation on the differentiation propensity of ESCs. We found an increased expression of trophectoderm-related genes including Cdx2 in Smarcb1-deficient ESCs. Consistently, they exhibited an extended differentiation propensity into the trophectoderm lineage cells in teratomas. However, although Smarcb1-deficient cells were infrequently incorporated into the trophectoderm cell layer of blastocysts, they failed to contribute to mature placental tissues in vivo. Furthermore, Smarcb1-deficient cells exhibited a premature differentiation in the neural tissue of E14.5 chimeric embryos. Notably, we found that binding motifs for CTCF, which is involved in the maintenance of genomic DNA architecture was significantly enriched in chromatin regions whose accessibility was augmented in Smarcb1-deficient cells, while those for pluripotency factors were overrepresented in regions which have more closed structure in those cells. Collectively, we propose that SMARCB1-mediated remodeling of chromatin landscapes is important for the maintenance and differentiation of ESCs.