The rBC2LCN-positive subpopulation of PC-3 cells exhibits cancer stem-like properties.

The recombinant lectin rBC2LCN is a useful marker for discriminating the undifferentiated status of human induced or embryonic stem cells. Recently, rBC2LCN has also been used for detecting some cancers and niche cells. However, the generality of which types of cells are detected by rBC2LCN is unclear. In this study, we demonstrated the potential of rBC2LCN as a probe for detecting and isolating cancer stem-like cells. Interestingly, flow cytometric analysis of various human cell lines indicated that the human prostate cancer cell line PC-3 consisted of rBC2LCN-positive and -negative subpopulations. Compared with the rBC2LCN-negative subpopulation, the rBC2LCN-positive subpopulation possessed representative features of cancer stem cells and malignancy, such as slow proliferation, increased cell motility, anchorage-independent growth, and drug resistance. The comprehensive expression profiles revealed that the rBC2LCN-positive subpopulation expressed higher levels of cancer stem cell markers. These findings indicate that rBC2LCN is useful for detecting not only pluripotent stem cells but also the cancer stem-like subpopulation of PC-3 cells. Pluripotent and cancer cells with rBC2LCN positivity would be important for future stem cell research.

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.

Structural and quantitative evidence for dynamic glycome shift on production of induced pluripotent stem cells.

We recently reported that induced pluripotent stem cells (iPSCs) prepared from different human origins acquired similar glycan profiles to one another as well as to human embryonic stem cells. Although the results strongly suggested attainment of specific glycan expressions associated with the acquisition of pluripotency, the detailed glycan structures remained to be elucidated. Here, we perform a quantitative glycome analysis targeting both N- and O-linked glycans derived from 201B7 human iPSCs and human dermal fibroblasts as undifferentiated and differentiated cells, respectively. Overall, the fractions of high mannose-type N-linked glycans were significantly increased upon induction of pluripotency. Moreover, it became evident that the type of linkage of Sia on N-linked glycans was dramatically changed from α-2-3 to α-2-6, and the expression of α-1-2 fucose and type 1 LacNAc structures became clearly apparent, while no such glycan epitopes were detected in fibroblasts. The expression profiles of relevant glycosyltransferase genes were fully consistent with these results. These observations indicate unambiguously the manifestation of a "glycome shift" upon conversion to iPSCs, which may not merely be the result of the initialization of gene expression, but could be involved in a more aggressive manner either in the acquisition or maintenance of the undifferentiated state of iPSCs.

rBC2LCN, a new probe for live cell imaging of human pluripotent stem cells.

Cell surface biomarkers have been applied to discriminate pluripotent human embryonic stem cells and induced pluripotent stem cells from differentiated cells. Here, we demonstrate that a recombinant lectin probe, rBC2LCN, a new tool for fluorescence-based imaging and flow cytometry analysis of pluripotent stem cells, is an alternative to conventional pluripotent maker antibodies. Live or fixed colonies of both human embryonic stem cells and induced pluripotent stem cells were visualized in culture medium containing fluorescent dye-labeled rBC2LCN. Fluorescent dye-labeled rBC2LCN was also successfully used to separate live pluripotent stem cells from a mixed cell population by flow cytometry.

Glycome diagnosis of human induced pluripotent stem cells using lectin microarray.

Induced pluripotent stem cells (iPSCs) can now be produced from various somatic cell (SC) lines by ectopic expression of the four transcription factors. Although the procedure has been demonstrated to induce global change in gene and microRNA expressions and even epigenetic modification, it remains largely unknown how this transcription factor-induced reprogramming affects the total glycan repertoire expressed on the cells. Here we performed a comprehensive glycan analysis using 114 types of human iPSCs generated from five different SCs and compared their glycomes with those of human embryonic stem cells (ESCs; nine cell types) using a high density lectin microarray. In unsupervised cluster analysis of the results obtained by lectin microarray, both undifferentiated iPSCs and ESCs were clustered as one large group. However, they were clearly separated from the group of differentiated SCs, whereas all of the four SCs had apparently distinct glycome profiles from one another, demonstrating that SCs with originally distinct glycan profiles have acquired those similar to ESCs upon induction of pluripotency. Thirty-eight lectins discriminating between SCs and iPSCs/ESCs were statistically selected, and characteristic features of the pluripotent state were then obtained at the level of the cellular glycome. The expression profiles of relevant glycosyltransferase genes agreed well with the results obtained by lectin microarray. Among the 38 lectins, rBC2LCN was found to detect only undifferentiated iPSCs/ESCs and not differentiated SCs. Hence, the high density lectin microarray has proved to be valid for not only comprehensive analysis of glycans but also diagnosis of stem cells under the concept of the cellular glycome.

rBC2LCN lectin as a potential probe of early-stage HER2-positive breast carcinoma.

The recombinant N-terminal domain of BC2L-C lectin (rBC2LCN) is useful for detecting not only human pluripotent stem cells but also some cancers. However, the cancer types and stages that can be detected by rBC2LCN remain unclear. In this study, we identified the human breast carcinoma subtypes and stages that can be detected by rBC2LCN. Compared with rBC2LCN-negative breast carcinoma cell lines, the rBC2LCN-positive cells expressed higher levels of human epidermal growth factor receptor 2 (HER2) and epithelial marker genes. Importantly, rBC2LCN histochemical staining of human breast carcinoma tissues demonstrated the utility of rBC2LCN in detecting breast carcinoma types that express HER2 and have not spread much in the early phase of growth. We conclude that rBC2LCN may have potential as a detection probe and a drug delivery vehicle to identify and treat early-stage HER2-positive breast carcinoma.

A technique for removing tumourigenic pluripotent stem cells using rBC2LCN lectin.

INTRODUCTION: Tumourigenesis attributed to residual undifferentiated cells in a graft is considered to be a significant issue in cell therapy using human pluripotent stem cells. To ensure the safety of regenerative medicine derived from pluripotent stem cells, residual undifferentiated cells must be eliminated in the manufacturing process. We previously described the lectin probe rBC2LCN, which binds harmlessly and specifically to the cell surface of human pluripotent stem cells. We report here a technique using rBC2LCN to remove pluripotent cells from a heterogenous population to reduce the chance of teratoma formation. METHODS: We demonstrate a method for separating residual tumourigenic cells using rBC2LCN-bound magnetic beads. This technology is a novel use of their previous discovery that rBC2LCN is a lectin that selectively binds to pluripotent cells. We optimize and validate a method to remove hPSCs from a mixture with human fibroblasts using rBC2LCN-conjugated magnetic beads. RESULTS: Cells with the potential to form teratoma could be effectively eliminated from a heterogeneous cell population with biotin-labelled rBC2LCN and streptavidin-bound magnetic beads. The efficiency was measured by FACS, ddPCR, and animal transplantation, suggesting that magnetic cell separation using rBC2LCN is quite efficient for eliminating hPSCs from mixed cell populations. CONCLUSIONS: The removal of residual tumourigenic cells based on rBC2LCN could be a practical option for laboratory use and industrialisation of regenerative medicine using human pluripotent stem cells.

Application of a human mesoderm tissue elongation system in vitro derived from human induced pluripotent stem cells to risk assessment for teratogenic chemicals.

Toxic compounds from the mother's diet and medication in addition to genetic factors and infection during pregnancy remain risks for various congenital disorders and misbirth. To ensure the safety of food and drugs for pregnant women, establishment of an in vitro system that morphologically resembles human tissues has been long desired. In this study, we focused on dorsal mesoderm elongation, one of the critical early development events for trunk formation, and we established in vitro autonomous elongating tissues from human induced pluripotent stem cells (hiPSCs). This artificial tissue elongation is regulated by MYOSIN II and FGF signaling, and is diminished by methylmercury or retinoic acid (RA), similar to in vivo human developmental disabilities. Moreover, our method for differentiation of hiPSCs requires only a short culture period, and the elongation is cell number-independent. Therefore, our in vitro human tissue elongation system is a potential tool for risk assessment assays for identification of teratogenic chemicals via human tissue morphogenesis.

Bestrophin genes are expressed in Xenopus development.

The Bestrophin-1/VMD2 gene has been implicated in Best disease, a juvenile-onset vitelliform macular dystrophy. The Bestrophin proteins have anion channel activity, and the four mammalian members share sequence homologies in multiple transmembrane domains and an RFP-tripeptide motif. The expression patterns and functions of the Bestrophin genes in retinal pigment epithelium have been studied intensively, whereas little is known about their roles in vertebrate embryogenesis. This study examined the roles of four Xenopus tropicalis homologs of BEST genes. The xtBest genes showed spatially and temporally distinct expression. xtBest-2 was the only maternally expressed Best gene, and both xtBest-2 and the Xenopus laevis Best-2 gene were expressed at the edge of the blastopore lip including the organizer. Ectopic expression of xBest-2 caused defects in dorsal axis formation and in mesodermal gene expression during gastrulation. These results suggest a new role of the Bestrophin family genes in early vertebrate embryogenesis.

Template Activating Factor-I α Regulates Retroviral Silencing during Reprogramming.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) is accompanied by dramatic changes in epigenetic programs, including silencing of endogenous and exogenous retroviruses. Here, we utilized replication-defective and persistent Sendai virus (SeVdp)-based vectors to monitor retroviral silencing during reprogramming. We observed that retroviral silencing occurred at an early reprogramming stage without a requirement for KLF4 or the YY1-binding site in the retroviral genome. Insertional chromatin immunoprecipitation (iChIP) enabled us to isolate factors assembled on the silenced provirus, including components of inhibitor of histone acetyltransferase (INHAT), which includes the SET/TAF-I oncoprotein. Knockdown of SET/TAF-I in mouse embryonic fibroblasts (MEFs) diminished retroviral silencing during reprogramming, and overexpression of template activating factor-I α (TAF-Iα), a SET/TAF-I isoform predominant in embryonic stem cells (ESCs), reinforced retroviral silencing by an SeVdp-based vector that is otherwise defective in retroviral silencing. Our results indicate an important role for TAF-Iα in retroviral silencing during reprogramming.

TRIQK, a novel family of small proteins localized to the endoplasmic reticulum membrane, is conserved across vertebrates.

Here we report a novel small protein that is highly conserved across vertebrates. The protein, which we have named TRIQK, has no homology to any previously reported proteins or functional domains, but all vertebrate homologs of this protein share a characteristic triple repeat of the sequence QXXK/R, as well as a hydrophobic C-terminal region. The Xenopus triqk gene (xTriqk) was isolated in an expression screen on the basis of its ability to cause dramatic changes in cell size and nuclear size and morphology in developing embryos. The Xenopus and mouse triqk genes are broadly expressed throughout embryogenesis, and mtriqk is also generally expressed in mouse adult tissues. TRIQK proteins are localized to the endoplasmic reticulum membrane. Depletion of endogenous xTRIQK protein in Xenopus embryos causes no detectable morphological or functional changes in tadpoles.

The N-terminus zinc finger domain of Xenopus SIP1 is important for neural induction, but not for suppression of Xbra expression.

Smad-interacting protein-1 (SIP1), also known as deltaEF2, ZEB2 and zfhx1b, is essential for the formation of the neural tube and the somites. Overexpression of Xenopus SIP1 causes ectopic neural induction via inhibition of bone morphogenetic protein (BMP) signaling and inhibition of Xbra expression. Here, we report the functional analyses of 4 domain-deletion mutants of XSIP1. Deletion of the N-terminus zinc finger domain suppressed neural induction and BMP inhibition, but these were not affected by deletion of the other domains (the Smad binding domain, the DNA-binding homeodomain together with the CtBP binding site and the C-terminus zinc finger). Therefore SIP1 does not inhibit BMP signaling by binding to Smad proteins. In contrast, all of the deletion constructs inhibited Xbra expression. These results suggest that the N-terminus zinc finger domain of XSIP1 has an important role in neural induction and that Xbra suppression occurs via a mechanism separate from the neural inducing activity.

A Serpin family gene, protease nexin-1 has an activity distinct from protease inhibition in early Xenopus embryos.

Protease nexin-1 (PN-1)/glia-derived nexin (GDN) is a member of the Serpin (serine proteinase inhibitor) family, and can inhibit thrombin, plasmin, and plasminogen activators. PN-1 has been shown to be a neuroprotective factor in a number of assay systems, and this activity has been assumed to be a function of its protease inhibitory function. Here, we report cloning and characterization of a Xenopus orthologue of PN-1 (xPN-1). xPN-1 was isolated in a functional screen of an egg cDNA library for factors that modify early axial patterning. xPN-1 is expressed maternally through late tadpole stages, and is expressed preferentially in the notochord, the pharyngeal endoderm, the otic vesicle, and the ventral region of the brain in tailbud embryos. Over-expression of xPN-1 causes defective gastrulation, inhibits convergent extension movements in activin induced animal caps, and inhibits expression of a distinct subset of activin induced mesendodermal markers. Interestingly, expression of point or deletion mutation of the Reactive Center Loop of xPN1,which is essential for the protease inhibitory activity of all serpins, had effects on Xenopus development indistinguishable from those of wild type xPN-1. These observations suggest the possibility that xPN-1 has a novel activity in addition to its established function as an inhibitor of serine proteases.

High hydrostatic pressure induces pro-osteoarthritic changes in cartilage precursor cells: A transcriptome analysis.

Due to the high water content of cartilage, hydrostatic pressure is likely one of the main physical stimuli sensed by chondrocytes. Whereas, in the physiological range (0 to around 10 MPa), hydrostatic pressure exerts mostly pro-chondrogenic effects in chondrocyte models, excessive pressures have been reported to induce detrimental effects on cartilage, such as increased apoptosis and inflammation, and decreased cartilage marker expression. Though some genes modulated by high pressure have been identified, the effects of high pressure on the global gene expression pattern have still not been investigated. In this study, using microarray technology and real-time PCR validation, we analyzed the transcriptome of ATDC5 chondrocyte progenitors submitted to a continuous pressure of 25 MPa for up to 24 h. Several hundreds of genes were found to be modulated by pressure, including some not previously known to be mechano-sensitive. High pressure markedly increased the expression of stress-related genes, apoptosis-related genes and decreased that of cartilage matrix genes. Furthermore, a large set of genes involved in the progression of osteoarthritis were also induced by high pressure, suggesting that hydrostatic pressure could partly mimic in vitro some of the genetic alterations occurring in osteoarthritis.

Development of a practical sandwich assay to detect human pluripotent stem cells using cell culture media.

Human pluripotent stem cells are considered to be ideal cell sources for regenerative medicine, but their clinical and industrial application is hindered by their tumorigenic potential. Previously we have identified a pluripotent stem cell-specific lectin rBC2LCN recognizing podocalyxin as a cell surface ligand. More recently, podocalyxin was found to be a soluble ligand of rBC2LCN that is secreted specifically from human pluripotent stem cells into cell culture media. Taking advantage of this phenomenon, we have previously developed a sandwich assay targeting the soluble podocalyxin using rBC2LCN as a capturing probe and another lectin rABA as an overlay probe to detect human pluripotent stem cells residing in cell therapy products derived from human pluripotent stem cells. A drawback to this, however, was that cell culture media containing fetal bovine serum was found to cause a substantial background signal to the sandwich assay. To reduce the background and increase the sensitivity, we screened different overlay probes to detect the soluble podocalyxin. Among them, an anti-keratan sulfate monoclonal antibody called R-10G showed the highest sensitivity and provided a low background signal to fetal bovine serum. The established sandwich assay using rBC2LCN and R-10G was proved to be powerful, which allowed the high-sensitive detection of human induced pluripotent stem cells residing among clinical-grade cardiomyocytes and neural stem cells, both derived from human induced pluripotent stem cells. The developed method has a possibility to be a standard technology to detect human induced pluripotent stem cells resided in various types of cell therapy products.

A Novel Probe as Surface Glycan Marker of Pluripotent Stem Cells: Research Outcomes and Application to Regenerative Medicine.

Human pluripotent stem cells (hPSCs), represented by embryonic stem (hESCs) and induced pluripotent stem cells (hiPSCs), are attracting increasing attention in various research fields. However, their application in a clinical scenario must overcome an important hurdle given that these cells are potentially tumorigenic. This inherent problem becomes more significant as the number of transplanted cells becomes larger. In this Progress Report, recent findings concerning a novel glycan marker for hPSCs are described, as well as attempts made in relation to its practical application to regenerative medicine. In line with current thinking in the glycoscience field, it is assumed that cellular glycomes are closely related to cell functions. Based on this premise, hESCs and hiPSCs are analyzed by an advanced glycan profiling technology--the high-density lectin microarray. It is found that all human iPSCs derived from different tissular origins show essentially the same glycan profiles, which are typified by several characteristic structural features. In addition, a recombinant lectin probe, rBC2LCN, which shows rigorous specificity to H type 1 and 3 glycan structures, is found to serve as an excellent probe for hPSCs.

Insulin-like factor regulates neural induction through an IGF1 receptor-independent mechanism.

Insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) signalling is required for normal embryonic growth and development. Previous reports indicated that the IGF/IGF1R/MAPK pathway contributes to neural induction and the IGF/IGF1R/PI3K/Akt pathway to eye development. Here, we report the isolation of insulin3 encoding a novel insulin-like ligand involved in neural induction. Insulin3 has a similar structure to pro-insulin and mature IGF ligands, but cannot activate the IGF1 receptor. However, similar to IGFs, Insulin3 induced the gene expression of an anterior neural marker, otx2, and enlarged anterior head structures by inhibiting Wnt signalling. Insulin3 are predominantly localised to the endoplasmic reticulum when otx2 is induced by insulin3. Insulin3 reduced extracellular Wnts and cell surface localised Lrp6. These results suggest that Insulin3 is a novel cell-autonomous inhibitor of Wnt signalling. This study provides the first evidence that an insulin-like factor regulates neural induction through an IGF1R-independent mechanism.

Elimination of tumorigenic human pluripotent stem cells by a recombinant lectin-toxin fusion protein.

The application of stem-cell-based therapies in regenerative medicine is hindered by the tumorigenic potential of residual human pluripotent stem cells. Previously, we identified a human pluripotent stem-cell-specific lectin probe, called rBC2LCN, by comprehensive glycome analysis using high-density lectin microarrays. Here we developed a recombinant lectin-toxin fusion protein of rBC2LCN with a catalytic domain of Pseudomonas aeruginosa exotoxin A, termed rBC2LCN-PE23, which could be expressed as a soluble form from the cytoplasm of Escherichia coli and purified to homogeneity by one-step affinity chromatography. rBC2LCN-PE23 bound to human pluripotent stem cells, followed by its internalization, allowing intracellular delivery of a cargo of cytotoxic protein. The addition of rBC2LCN-PE23 to the culture medium was sufficient to completely eliminate human pluripotent stem cells. Thus, rBC2LCN-PE23 has the potential to contribute to the safety of stem-cell-based therapies.

A stable chimeric fibroblast growth factor (FGF) can successfully replace basic FGF in human pluripotent stem cell culture.

Fibroblast growth factors (FGFs) are essential for maintaining self-renewal in human embryonic stem cells and induced pluripotent stem cells. Recombinant basic FGF (bFGF or FGF2) is conventionally used to culture pluripotent stem cells; however, because of the instability of bFGF, repeated addition of fresh bFGF into the culture medium is required in order to maintain its concentration. In this study, we demonstrate that a heat-stable chimeric variant of FGF, termed FGFC, can be successfully used for maintaining human pluripotent stem cells. FGFC is a chimeric protein composed of human FGF1 and FGF2 domains that exhibits higher thermal stability and protease resistance than do both FGF1 and FGF2. Both human embryonic stem cells and induced pluripotent stem cells were maintained in ordinary culture medium containing FGFC instead of FGF2. Comparison of cells grown in FGFC with those grown in conventional FGF2 media showed no significant differences in terms of the expression of pluripotency markers, global gene expression, karyotype, or differentiation potential in the three germ lineages. We therefore propose that FGFC may be an effective alternative to FGF2, for maintenance of human pluripotent stem cells.

Live-cell imaging of human pluripotent stem cells by a novel lectin probe rBC2LCN.

We performed comprehensive glycome analysis of a large set of human pluripotent stem cells (hPSCs) using a high-density lectin microarray. We found that a recombinant lectin, rBC2LCN, binds exclusively to all of the undifferentiated hPSCs tested, but not to differentiated somatic cells. rBC2LCN can be used for both the staining and sorting of fixed and live hPSCs. rBC2LCN could serve as a novel detection reagent for hPSCs, particularly given that rBC2LCN is cost effective and, unlike conventional antibodies which require mammalian cells for their production, is easy to produce in a large amount (0.1 g/L) in an Escherichia coli expression system. Here we describe protocols for the fluorescence staining of fixed and live hPSCs and their detection by flow cytometry.