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Cancer stem cells are a promising target for cancer eradication due to their responsibility for therapy-resistance and cancer recurrence. Previously, we have demonstrated that glioma stem cells (GSCs) recruit and induce the differentiation of bone marrow (BM) monocytes into tumor-infiltrating macrophages, which phagocytose hemorrhaged erythrocytes and store GSC-beneficial iron in mouse xenografts, suggesting a self-expanding strategy of GSCs that exploits host hematopoiesis of myeloid cells. However, it remains unclear whether a self-advantageous effect of GSCs also occurs on erythroid cells during glioma development. Here, we found that, in the primary cultures of mouse fetal liver proerythroblasts (proEs), conditioned media prepared from glioma cells including patient-derived glioblastoma (GBM) cells significantly facilitated the differentiation of proEs into erythroblasts. Importantly, in-vivo erythroid analysis in intracranially GSC-transplanted mice showed an enhanced erythropoiesis in the BM. In addition, the sphere forming ability of patient-derived GBM cells was significantly suppressed by hypoxia treatment and iron chelation, suggesting higher demands of GSCs for oxygen and iron, which may be supplied by GSCs- and their progeny-induced erythrocyte production. Our findings provide a new insight into survival and expanding strategies of GSCs that systemically exploit host erythropoiesis.

BackgroundDuring mouse embryonic development, definitive hematopoiesis is first detected around embryonic day (E) 10.5 in the aorta-gonad-mesonephros (AGM) region. Hematopoietic stem cells (HSCs) arise in the dorsal aorta’s intra-aortic hematopoietic cell clusters (IAHCs). We have previously reported that a transcription factor Sox17, is expressed in IAHCs, and that, among them, CD45lowc-Kithighcells have high hematopoietic activity. Furthermore, forced expression of Sox17 in this population of cells can maintain the formation of hematopoietic cell clusters. However, how Sox17 does so, particularly downstream signaling involved, remains poorly understood. The purpose of this study is to search for new Sox17 targets which contribute to cluster formation with hematopoietic activity.MethodsRNA-sequencing (RNA-seq) analysis was done to identify genes that are up- regulated in Sox17-expressing IAHCs as compared with Sox17-negative ones. Among the top 7 highly expressed genes, Rasip1 which had been reported to be a vascular-specific regulator was focused on in this study and firstly the whole-mount immunostaining was done. We conducted a luciferase reporter assay to identify the Sox17 binding site for Rasip1 gene induction. We also analyzed the cluster formation and the multi-lineage colony forming ability of Rasip1-transduced cells and Rasip1-knockdown Sox17-transduced cells.ResultsThe increase of theRasip1expression level was observed in Sox17-positive CD45lowc-Kithighcells as compared with the Sox17-nonexpressing control. Also, the expression level of theRasip1gene was increased by the Sox17-nuclear translocation. Rasip1 was expressed on the membrane of IAHCs, overlapping with the endothelial cell marker, CD31, and hematopoietic stem/progenitor marker (HSPC), c-Kit. Overexpression of Rasip1 in CD45lowc-Kithighcells led to a significant but transient increase in hematopoietic activity, while Rasip1 knock-down in Sox17-transduced cells decreased the cluster formation and diminished the colony-forming ability.ConclusionsRasip1 knockdown in Sox17-transduced CD45lowc-Kithighcells displayed a significant decrease in the multi-lineage colony forming ability and the cluster size. Rasip1 overexpression in Sox17-untransduced CD45lowc-Kithighcells led to a significant but transient increase in the multi-lineage colony forming ability, suggesting the presence of a cooperating factor for sustained hematopoietic activity.

Hematopoietic stem cell-containing intra-aortic hematopoietic cell clusters (IAHCs) emerge in the dorsal aorta of the aorta-gonad-mesonephros (AGM) region during midgestation mouse embryos. We previously showed that transduction of Sox17 in CD45lowc-Kithigh cells, which are one component of IAHCs, maintained the cluster formation and the undifferentiated state, but the mechanism of the cluster formation by Sox17 has not been clarified. By microarray gene expression analysis, we found that genes for vascular endothelial-cadherin (VE-cad) and endothelial cell-selective adhesion molecule (ESAM) were expressed at high levels in Sox17-transduced c-Kit+ cells. Here we show the functional role of these adhesion molecules in the formation of IAHCs and the maintenance of the undifferentiated state by in vitro experiments. We detected VE-cad and ESAM expression in endothelial cells of dorsal aorta and IAHCs in E10.5 embryos by whole mount immunohistochemistry. Cells with the middle expression level of VE-cad and the low expression level of ESAM had the highest colony-forming ability. Tamoxifen-dependent nuclear translocation of Sox17-ERT fusion protein induced the formation of cell clusters and the expression of Cdh5 (VE-cad) and ESAM genes. We showed the induction of the Cdh5 (VE-cad) and ESAM expression and the direct interaction of Sox17 with their promoter by luciferase assay and chromatin immunoprecipitation assay, respectively. Moreover, shRNA-mediated knockdown of either Cdh5 (VE-cad) or ESAM gene in Sox17-transduced cells decreased the multilineage-colony forming potential. These findings suggest that VE-cad and ESAM play an important role in the high hematopoietic activity of IAHCs and cluster formation.

Y-box binding protein 2 (YBX2) has been associated with the properties of both germ cells and cancer cells. We hypothesized that YBX2 might contribute to the characteristics of cancer stem cells (CSCs). In this study, we clarified the function of YBX2 in endometrial cancer stem cells. We established a human YBX2-expressing Ishikawa (IK) cell line (IK-YBX2 cells). We analyzed gene expression associated with stemness and isolated SP cells from IK-YBX2 cells. The SP population of IK-YBX2 cells, the expression of ALDH1 and serial sphere-forming capacity were associated with levels of YBX2 expression. IK-YBX2 cells were resistant to anti-cancer drugs. In gene expression analysis, a gene for cancer testis antigen, CT45, was generally overexpressed in IK-YBX2 cells. YBX2-mediated CT45 expression was associated with increased levels of self-renewal capacity and paclitaxel resistance. The level of CT45 expression was enhanced in high-grade and/or advanced stages of human endometrial cancer tissues. We conclude that expression of YBX2 is essential for the stem cell-like phenotype. CT45 contributes to stemness associated with YBX2 and might be related to the progression of endometrial cancer.

A transcription factor Sry-related high mobility group box (Sox) 17 is involved in developmental processes including spermatogenesis, cardiovascular system, endoderm formation, and so on. In this article, we firstly review the studies on the relation between the Sox17 expression and tumor malignancy. Although Sox17 positively promotes various tissue development, most of the cancers associated with Sox17 show decreased expression levels of Sox17, and an inverse correlation between Sox17 expression and malignancy is revealed. We briefly discuss the mechanism of such Sox17 down-regulation by focusing on DNA methylation of CpG sites located in the Sox17 gene promoter. Next, we overview the function of Sox17 in the fetal hematopoiesis, particularly in the dorsal aorta in midgestation mouse embryos. The Sox17 expression in hematopoietic stem cell (HSC)-containing intra-aortic hematopoietic cell cluster (IAHCs) is important for the cluster formation with the hematopoietic ability. The sustained expression of Sox17 in adult bone marrow HSCs and the cells in IAHCs of the dorsal aorta indicate abnormalities that are low lymphocyte chimerism and the aberrant proliferation of common myeloid progenitors in transplantation experiments. We then summarize the perspectives of Sox17 research in cancer control.

Due to the nature of enhanced resistance to conventional chemo/radiotherapies and metastasis, highly tumorigenic cancer stem cells (CSCs) have been proposed as a promising target for cancer eradication. To tackle the therapeutic difficulties of cancers involving CSCs, extensive research efforts have been directed toward understanding the extracellular microenvironments of CSCs, i.e., CSC niche, which plays important roles in CSC maintenance and expansion. Here we review recently identified mechanisms of maintenance and expansion of glioma CSCs (GSCs) leading to glioma progression and recurrence, with particular emphasis on the reports made by studies with a unique approach using polymer microarrays screening and with a unique viewpoint of necrotic particles. The polymer-based approach identified two groups of niche components, extracellular matrices (ECMs) and iron, and uncovered that co-expression of ECM-, iron-, and macrophage-related genes is predictive of glioma patients’ outcome. The study in view of a unique fraction of GSC-derived necrotic particles proposed that such particles develop GSC-supportive tumor-associated macrophages (TAMs). Taken together, these studies provide new insights into the mechanisms underlying GSC-driven niche development, i.e., organization of the self-advantageous niche microenvironments for GSC maintenance and expansion leading to glioma progression and recurrence. A series of such studies can redefine the current concept of anti-GSC niche therapy that targets ligands/receptors supporting GSCs, and have potential to accelerate cancer therapy development.

In the midgestation mouse embryo, hematopoietic cell clusters containing hematopoietic stem/progenitor cells arise in the aorta-gonad-mesonephros (AGM) region. We have previously reported that forced expression of the Sox17 transcription factor in CD45lowc-Kithigh AGM cells, which are the hematopoietic cellular component of the cell clusters, and subsequent coculture with OP9 stromal cells in the presence of three cytokines, stem cell factor (SCF), interleukin-3 (IL-3), and thrombopoietin (TPO), led to the formation and the maintenance of cell clusters with cells at an undifferentiated state in vitro. In this study, we investigated the role of each cytokine in the formation of hematopoietic cell clusters. We cultured Sox17-transduced AGM cells with each of the 7 possible combinations of the three cytokines. The size and the number of Sox17-transduced cell clusters in the presence of TPO, either alone or in combination, were comparable to that observed with the complete set of the three cytokines. Expression of TPO receptor, c-Mpl was almost ubiquitously expressed and maintained in Sox17-transduced hematopoietic cell clusters. In addition, the expression level of c-Mpl was highest in the CD45lowc-Kithigh cells among the Sox17-transduced cell clusters. Moreover, c-Mpl protein was highly expressed in the intra-aortic hematopoietic cell clusters in comparison with endothelial cells of dorsal aorta. Finally, stimulation of the endothelial cells prepared from the AGM region by TPO induced the production of hematopoietic cells. These results suggest that TPO contributes to the formation and the maintenance of hematopoietic cell clusters in the AGM region.

Spontaneous necrosis is a defining feature of glioblastomas (GBMs), the most malignant glioma. Despite its strong correlations with poor prognosis, it remains unclear whether necrosis could be a possible cause or mere consequence of glioma progression. Here we isolated a particular fraction of necrotic products spontaneously arising from glioma cells, morphologically and biochemically defined as autoschizis-like products (ALPs). When administered to granulocyte macrophage colony-stimulating factor (GM-CSF)-primed bone marrow-derived macrophage/dendritic cells (Mφ/DCs), ALPs were found to be specifically engulfed by Mφs expressing a tumor-associated macrophage (TAM) marker CD204. ALPs from glioma stem cells (GSCs) had higher activity for the TAM development than those from non-GSCs. Of note, expression of the Il12b gene encoding a common subunit of IL-12/23 was upregulated in ALPs-educated Mφs. Furthermore, IL-12 protein evidently enhanced the sphere-forming activity of GBM patient-derived cells, although interestingly IL-12 is generally recognized as an antitumoral M1-Mφ marker. Finally, in silico analysis of The Cancer Genome Atlas (TCGA) transcriptome data of primary and recurrent GBMs revealed that higher expression of these IL-12 family genes was well correlated with more infiltration of M1-type TAMs and closely associated with poorer prognosis in recurrent GBMs. Our results highlight a role of necrosis in GSC-driven self-beneficial niche construction and glioma progression, providing important clues for developing new therapeutic strategies against gliomas.

Cancer stem cells (CSCs) are maintained under special microenvironment called niche, and elucidation and targeting of the CSC niche will be a feasible strategy for cancer eradication. Tumor-associated macrophages (TAMs) are known to be involved in cancer progression and thus can be a component of CSC niche. Although TAMs are known to play multiple roles in tumor progression, involvement of CSCs in TAM development fully remains to be elucidated. Using rat C6 glioma side population (SP) cells as a model of glioma CSCs, we here show that CSCs induce the TAM development by promoting survival and differentiation of bone marrow-derived monocytes. CSC-induced macrophages can be separated into two distinct subsets of cells, CD11c(low) and CD11c(high) cells. Interestingly, only the CD11c(high) subset of cells have protumoral activity, as shown by intracranial transplantation into immune-deficient mice together with CSCs. These CD11c(high) macrophages were observed in the tumor formed by co-transplantation with CSCs. Furthermore, CSCs produced GM-CSF and anti-GM-CSF antibody inhibited CSC-induced TAM development. In conclusion, CSCs have the ability to self-create their own niche involving TAMs through CSC-derived GM-CSF, which can thus be a therapeutic target in view of CSC niche disruption.

GASC1, also known as KDM4C/JMJD2C, is a histone demethylase for histone H3 lysine 9 (H3K9) and H3K36. In this study, we observed an increase of GFAP-positive astrocytes in the brain of Gasc1 hypomorphic mutant mice at 2-3 months of age, but not at postnatal day 14 and day 30 by immunohistochemistry. Increases of GFAP-positive astrocytes were widely observed in the forebrain and prominent in such regions as cerebral cortex, caudate putamen, amygdala and diencephalon, but not obvious in hippocampus. Taken together with our observations to be published elsewhere that Gasc1 hypomorphic mutant mice exhibit abnormal behaviors including hyperactivity, persistence and many types of learning and memory deficits and abnormal synaptic functions such as prolonged long-term potentiation, the increase in GFAP-positive astrocytes may help understand their phenotypes, because astrocytes are known to affect synaptic plasticity.

Synovial sarcoma accounts for almost 10% of all soft tissue sarcomas, and its prognosis is poor with 5-year survival rates at 36%. Thus, new treatments and therapeutic targets for synovial sarcoma are required. Tumor-initiating cells have been defined by the ability for self-renewal and multipotent differentiation, and they exhibit higher tumorigenic capacity, chemoresistance and radiation resistance, expecting to be a new therapeutic target. In synovial sarcoma, the presence of such stemness remains largely unclear; thus, we analyzed whether synovial sarcoma possessed tumor-initiating cells and explored specific markers, and we discovered that synovial sarcoma cell lines possessed heterogeneity by way of containing a sphere-forming subpopulation highly expressing NANOG, OCT4 and SOX2. By expression microarray analysis, CXCR4 was identified to be highly expressed in the sphere subpopulation and correlated with stem-cell-associated markers. Inhibition of CXCR4 suppressed the cell proliferation of synovial sarcoma cell lines in vitro. The tumor-initiating ability of CXCR4-positive cells was demonstrated by xenograft propagation assay. CXCR4-positive cells showed higher tumorigenicity than negative ones and possessed both self-renewal and multipotent differentiation ability. Immunohistochemical analysis of 39 specimens of synovial sarcoma patients revealed that CXCR4 strongly correlated with poor prognosis of synovial sarcoma. Thus, we conclude that CXCR4 is the marker of synovial sarcoma-initiating cells, a new biomarker for prognosis and a new potential therapeutic target.

The aorta-gonad-mesonephros region, from which definitive hematopoiesis first arises in midgestation mouse embryos, has intra-aortic hematopoietic clusters (IAHCs) containing hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). We previously reported expression of the transcription factor Sox17 in IAHCs, and overexpression of Sox17 in CD45lowc-KIThigh cells comprising IAHCs maintains the formation of cell clusters and their multipotency in vitro over multiple passages. Here, we demonstrate the importance of NOTCH1 in IAHC formation and maintenance of the HSC/HPC phenotype. We further show that Notch1 expression is positively regulated by SOX17 via direct binding to its gene promoter. SOX17 and NOTCH1 were both found to be expressed in vivo in cells of IAHCs by whole mount immunostaining. We found that cells transduced with the active form of NOTCH1 or its downstream target, Hes1, maintained their multipotent colony-forming capacity in semisolid medium. Moreover, cells stimulated by NOTCH1 ligand, Jagged1, or Delta-like protein 1, had the capacity to form multilineage colonies. Conversely, knockdown of Notch1 and Hes1 led to a reduction of their multipotent colony-forming capacity. These results suggest that the Sox17-Notch1-Hes1 pathway is critical for maintaining the undifferentiated state of IAHCs.

The continual proliferation and differentiation of trophoblasts are critical for the maintenance of pregnancy. It is well known that the tissue stem cells are associated with the development of tissues and pathologies. It has been demonstrated that side-population (SP) cells identified by fluorescence-activated cell sorting (FACS) are enriched with stem cells. The SP cells in HTR-8/SVneo cells derived from human primary trophoblast cells were isolated by FACS. HTR-8/SVneo-SP cell cultures generated both SP and non-SP (NSP) subpopulations. In contrast, NSP cell cultures produced NSP cells and failed to produce SP cells. These SP cells showed self-renewal capability by serial colony-forming assay. Microarray expression analysis using a set of HTR-8/SVneo-SP and -NSP cells revealed that SP cells overexpressed several stemness genes including caudal type homeobox2 (CDX2) and bone morphogenic proteins (BMPs), and lymphocyte antigen 6 complex locus D (LY6D) gene was the most highly up-regulated in HTR-8/SVneo-SP cells. LY6D gene reduced its expression in the course of a 7-day cultivation in differentiation medium. SP cells tended to reduce its fraction by treatment of LY6D siRNA indicating that LY6D had potential to maintain cell proliferation of HTR-8/SVneo-SP cells. On ontology analysis, epithelial-mesenchymal transition (EMT) pathway was involved in the up-regulated genes on microarray analysis. HTR-SVneo-SP cells showed enhanced migration. This is the first report that LY6D was important for the maintenance of HTR-8/SVneo-SP cells. EMT was associated with the phenotype of these SP cells.

Cancer stem cells (CSCs) are dominantly responsible for tumor progression and chemo/radio-resistance, resulting in tumor recurrence. 5-aminolevulinic acid (ALA) is metabolized to fluorescent protoporphyrin IX (PpIX) specifically in tumor cells, and therefore clinically used as a reagent for photodynamic diagnosis (PDD) and therapy (PDT) of cancers including gliomas. However, it remains to be clarified whether this method could be effective for CSC detection. Here, using flow cytometry-based analysis, we show that side population (SP)-defined C6 glioma CSCs (GSCs) displayed much less 5-ALA-derived PpIX fluorescence than non-GSCs. Among the C6 GSCs, cells with ultralow PpIX fluorescence exhibited dramatically higher tumorigenicity when transplanted into the immune-deficient mouse brain. We further demonstrated that the low PpIX accumulation in the C6 GSCs was enhanced by deferoxamine (DFO)-mediated iron chelation, not by reserpine-mediated inhibition of PpIX-effluxing ABCG2. Finally, we found that the expression level of the gene for heme oxygenase-1 (HO-1), a heme degradation enzyme, was high in C6 GSCs, which was further up-regulated when treated with 5-ALA. Our results provide important new insights into 5-ALA-based PDD of gliomas, particularly photodetection of SP-defined GSCs by iron chelation based on their ALA-PpIX-Heme metabolism.

Sry related high mobility group box 17 (Sox17), which is a marker of endodermal cells and a transcriptional regulator, has a critical role in the maintenance of fetal and neonatal hematopoietic stem cells (HSC). Sox17 has been identified as a key regulator of the development and differentiation of fetal hematopoietic progenitors from the aorta-gonad-mesonephros (AGM) region. The co-culture of Sox17-transduced hematopoietic progenitor cells (CD45(low) c-Kit(high) cells) from AGM regions on OP9 stromal cells gives rise to multipotential hematopoietic stem/progenitor cells. Here, we show that in a primary transplantation experiment, Sox17-transduction in CD45(low) c-Kit(high) cells of embryonic day (E) 10.5 AGM increased the absolute number of common myeloid progenitors (CMPs) in the bone marrow (BM) of recipient mice in comparison to that of granulocyte/macrophage progenitors (GMPs) and the megakaryocyte/erythroid progenitors (MEPs). When Sox17-transduced cells were cultured with OP9 stromal cells, Sox17-transduced GMPs (Sox17-GMPs), Sox17-transduced CMPs (Sox17-CMPs), and Sox17-transduced MEPs (Sox17-MEPs) were generated. Sox17-GMPs and Sox17-CMPs maintained their self-renewal capacity and the hematopoietic ability upon co-culture with the OP9 stromal cells for some passages. Moreover, Sox17-GMPs exhibited the increase in expression of c-Mpl and GATA-2 in comparison to GMPs of BM and Sox17-CMPs showed the increase in expression of c-Mpl, NF-E2, and β-globin genes in comparison to CMPs of BM. Furthermore, when Sox17-transduced cells were cultured in methylcellulose to examine the colony-forming ability, Sox17-GMPs and Sox17-CMPs maintained the formation of mixed colonies for some passages. Taken together, Sox17 is suggested to regulate the maintenance and differentiation of hematopoietic progenitors derived from AGM regions at midgestation, in particular myeloid progenitors.

Goals We previously demonstrated that side-population (SP) cells in human endometrial cancer cells (Hec1 cells) and in rat endometrial cells expressing oncogenic human K-Ras protein (RK12V cells) have features of cancer stem cells (CSCs). Hec1-SP cells showed enhanced migration and the potential to differentiate into the mesenchymal cell lineage. In this study, we analyzed the association of the epithelial–mesenchymal transition (EMT) with the properties of these endometrial CSCs. We also assessed the effects of salinomycin (a compound with EMT-specific toxicity) on the proliferative capacity, migration and invasiveness of these endometrial CSCs using Hec1-SP cells. Method We performed microarray expression analysis to screen for up-regulated genes in CSCs using a set of RK12V-SP cells and -non-SP(NSP) cells and used the Metacore package to identify the Gene GO pathway MAPs involved in the up-regulated genes. To analyze their association with EMT, the expression of several EMT associated genes in Hec1-SP cells was investigated by real time PCR and compared with that in Hec1-NSP cells. We assessed the expression of BAX , BCL2 , LEF1 , cyclinD and fibronectin by real time PCR. We also evaluated the viabilities, migration and invasive activities, and tumorigenicities of these SP cells and NSP cells in the presence or absence of salinomycin. Results We demonstrated that i) EMT processes were observed in both RK12V-SP cells and Hec1-SP cells, ii) the level of fibronectin was enhanced in Hec1-SP cells and salinomycin reduced the level of fibronectin expression, iii) salinomycin induced apoptosis and inhibited Wnt signaling, and iv) salinomycin inhibited the proliferation, migration, invasiveness and tumorigenicity of these SP cells. Conclusion This is the first report of an inhibitory effect of salinomycin on the properties of endometrial CSCs.

Congenital biliary atresia is an incurable disease of newborn infants, of unknown genetic causes, that results in congenital deformation of the gallbladder and biliary duct system. Here, we show that during mouse organogenesis, insufficient SOX17 expression in the gallbladder and bile duct epithelia results in congenital biliary atresia and subsequent acute ‘embryonic hepatitis’, leading to perinatal death in ~95% of the Sox17 heterozygote neonates in C57BL/6 (B6) background mice. During gallbladder and bile duct development, Sox17 was expressed at the distal edge of the gallbladder primordium. In the Sox17+/− B6 embryos, gallbladder epithelia were hypoplastic, and some were detached from the luminal wall, leading to bile duct stenosis or atresia. The shredding of the gallbladder epithelia is probably caused by cell-autonomous defects in proliferation and maintenance of the Sox17+/− gallbladder/bile duct epithelia. Our results suggest that Sox17 plays a dosage-dependent function in the morphogenesis and maturation of gallbladder and bile duct epithelia during the late-organogenic stages, highlighting a novel entry point to the understanding of the etiology and pathogenesis of human congenital biliary atresia.

Background Elucidating the precise properties of cancer stem cells (CSCs) is indispensable for the development of effective therapies against tumors, because CSCs are key drivers of tumor development, metastasis and relapse. We previously reported that the Hoechst 33342 dye-low staining side population (SP) method can enrich for CSCs in the C6 glioma cell line, and that the positively stained main population (MP) cells are non-CSCs. Presence of cancer stem-like SP cells is reported in various types of cancer. Although altered cellular energy metabolism is a hallmark of cancer, very little has been studied on the applicability of fluorescent probes for the understanding of CSC energy metabolism. Methods The metabolic status of C6 SP and MP cells are evaluated by CellROX, MitoTracker Green (MTG) and JC-1 for cellular oxidative stress, mitochondrial amount, and mitochondrial membrane potential, respectively. Results SP cells were found to exhibit significantly lower fluorescent intensities of CellROX and MTG than MP cells. However, inhibition of ATP binding cassette (ABC) transporters by verapamil enhanced the intensities of these probes in SP cells to the levels similar to those in MP cells, indicating that SP cells expel the probes outside of the cells through ABC transporters. Next, SP cells were stained with JC-1 dye which exhibits membrane potential dependent accumulation in mitochondrial matrix, followed by formation of aggregates. The mitochondrial membrane potential indicated by the aggregates of JC-1 was 5.0-fold lower in SP cells than MP cells. Inhibition of ABC transporters enhanced the fluorescent intensities of the JC-1 aggregates in both SP and MP cells, the former of which was still 2.2-fold lower than the latter. This higher JC-1 signal in MP cells was further found to be due to the Hoechst 33342 dye existing in MP cells. When SP and MP cells were recultured to deprive the intracellular Hoechst 33342 dye and then stained with JC-1 in the presence of verapamil, the intensities of JC-1 aggregates in such SP and MP cells became comparable. Conclusion Inhibiting ABC transporters and depriving Hoechst 33342 dye are required for the accurate assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes.

During murine embryonic development, primitive hematopoiesis occurs in the yolk sac (YS). Recent studies have shown that the YS also harbors definitive hematopoietic activity. However, the population of YS cells contributing to definitive hematopoiesis has not been identified. In this study, we characterized the hematopoietic cell populations in the YS of mouse embryos from E9.5 to E14.5 in view of the expression profiles of CD45 and c-Kit. The YS cells from E9.5 to E11.5 could be divided into six populations: CD45−c-Kit−, CD45−c-Kitlow, CD45−c-Kithigh, CD45lowc-Kithigh, CD45highc-Kithigh and CD45highc-Kitvery low. Among these populations, CD45lowc-Kithigh cells showed the highest multilineage hematopoietic colony-forming activity. Later in development, the YS cells from E12.5 to E14.5 lost the second and fourth populations (i.e., they retained CD45−c-Kit−, CD45−c-Kithigh, CD45highc-Kithigh and CD45highc-Kitvery low cells), and concurrently with the disappearance of the CD45lowc-Kithigh population, no significant hematopoietic activity was found in any of the populations on and after E12.5. CD45lowc-Kithigh YS cells, which had a round morphology with a large nucleus, possessed the ability to differentiate into myeloid and B lymphoid cells when cultured with stromal cells. These findings suggest that CD45lowc-Kithigh YS cells include more undifferentiated cells than the other YS cell populations and possess in vitro potency to differentiate into multilineage hematopoietic cells. Furthermore, this cell population disappears from the YS at around E12.5, when the site of hematopoiesis has already shifted to the fetal liver and the placenta.

The discovery of a major hematopoietic stem cell pool in midgestation mouse embryo has defined the placenta as an important hematopoietic anatomical site. In this study, we examined the flow cytometric pattern of mouse placenta cells on embryonic days (E) 10.5 to E15.5, in view of CD45 and c-Kit expression. We also determined which population of these cells shows differentiation potential toward multiple hematopoietic lineages by performing coculture with OP9 stromal cells and colony-forming assay in methylcellulose. Only CD45+c-Kit+ population showed the ability to form hematopoietic colonies including multiple lineages. To distinguish which fraction of placenta cells have the hematopoietic activity, we used GFP transgenic mice in which the fetal part of the placenta is GFP positive and the maternal part is GFP negative. E11.5 and E13.5 CD45+c-Kit+ placental cells that have ability to form hematopoietic colonies are the fetal GFP positive placental cells. E11.5 and E13.5 CD45+c-Kit+ placental cells that have an ability to form hematopoietic colonies mainly reside in Hoechst dye-effluxing side population area (SP). Taken together, in the placenta of mouse embryo, we conclude that SP cells in the CD45+c-Kit+ fetal placental cells have the ability to form hematopoietic colonies.

Retinal astrocytes and their precursor cells migrate from the optic nerve. Interleukin 6 family cytokines, whose signal transduction requires gp130, promote astrocyte differentiation in the optic nerve, though the mechanism of astrocyte differentiation in the retina has not been clarified. We found that GFAP-positive astrocytes were significantly decreased in number but that a considerable number of astrocytes were still present in gp130-deficient mouse retina. These findings suggest that gp130-dependent signaling pathways play essential roles in retinal astrocyte differentiation and that retinal astrocyte differentiation can also be promoted by other signaling pathways. We found that leukemia inhibitory factor, bone morphogenetic proteins, and their receptors are expressed in P0 retina. In addition, leukemia inhibitory factor and bone morphogenetic protein 2 synergistically promote astrocyte differentiation of retinal precursor cells isolated from P0 mouse retina. These observations demonstrated that not only gp130-dependent signaling but also bone morphogenetic proteins play essential roles in retinal astrocyte differentiation.

Definitive hematopoiesis starts in the aorta-gonad-mesonephros (AGM) region of the mouse embryo. Our previous studies revealed that STAT3, a gp130 downstream transcription factor, is required for AGM hematopoiesis and that homeodomain-interacting protein kinase 2 (HIPK2) phosphorylates serine-727 of STAT3. HIPK2 is a serine/threonine kinase known to be involved in transcriptional repression and apoptosis. In the present study, we examined the role of HIPK2 in hematopoiesis in mouse embryo. HIPK2 transcripts were found in fetal hematopoietic tissues such as the mouse AGM region and fetal liver. In cultured AGM cells, HIPK2 protein was detected in adherent cells. Functional analyses of HIPK2 were carried out by introducing wild-type and mutant HIPK2 constructs into AGM cultures. Production of CD45(+) hematopoietic cells was suppressed by forced expression of HIPK2 in AGM cultures. This suppression required the kinase domain and nuclear localization signals of HIPK2, but the kinase activity was dispensable. HIPK2-overexpressing AGM-derived nonadherent cells did not form cobblestone-like colonies in cultures with stromal cells. Furthermore, overexpression of HIPK2 in AGM cultures impeded the expansion of CD45(low)c-Kit(+) cells, which exhibit the immature hematopoietic progenitor phenotype. These data indicate that HIPK2 plays a negative regulatory role in AGM hematopoiesis in the mouse embryo.

Cancer stem cells (CSCs) are believed to be maintained within a microenvironmental niche. Here we used polymer microarrays for the rapid and efficient identification of glioma CSC (GSC) niche mimicries and identified a urethane-based synthetic polymer, upon which two groups of niche components, namely extracellular matrices (ECMs) and iron are revealed. In cultures, side population (SP) cells, defined as GSCs in the rat C6 glioma cell line, are more efficiently sustained in the presence of their differentiated progenies expressing higher levels of ECMs and transferrin, while in xenografts, ECMs are supplied by the vascular endothelial cells (VECs), including SP cell-derived ones with distinctively greater ability to retain xenobiotics than host VECs. Iron is stored in tumor infiltrating host macrophages (Mφs), whose protumoral activity is potently enhanced by SP cell-secreted soluble factor(s). Finally, coexpression of ECM-, iron-, and Mφ-related genes is found to be predictive of glioma patients' outcome. Our polymer-based approach reveals the intrinsic capacities of GSCs, to adapt the environment to organize a self-advantageous microenvironment niche, for their maintenance and expansion, which redefines the current concept of anti-CSC niche therapy and has the potential to accelerate cancer therapy development.

Adult retinal stem cells represent a possible cell source for the treatment of retinal degeneration. However, only a small number of stem cells reside in the ciliary margin. The present study aimed to promote the proliferation of adult retinal stem cells via the Wnt signaling pathway. Ciliary margin cells from 8-week-old mice were dissociated and cultured to allow sphere colony formation. Wnt3a, a glycogen synthase kinase (GSK) 3 inhibitor, fibroblast growth factor (FGF) 2, and a FGF receptor inhibitor were then applied in the culture media. The primary spheres were dissociated to prepare either monolayer or secondary sphere cultures. Wnt3a increased the size of the primary spheres and the number of Ki-67–positive proliferating cells in monolayer culture. The Wnt3a-treated primary sphere cells were capable of self-renewal and gave rise to fourfold the number of secondary spheres compared with nontreated sphere cells. These cells also retained their multilineage potential to express several retinal markers under differentiating culture conditions. The Wnt3a-treated cells showed nuclear accumulation of β-catenin, and a GSK3 inhibitor, SB216763, mimicked the mitogenic activity of Wnt3a. The proliferative effect of SB216763 was attenuated by an FGF receptor inhibitor but was enhanced by FGF2, with Ki-67–positive cells reaching over 70% of the total cells. Wnt3a and SB216763 promoted the proliferation of retinal stem cells, and this was partly dependent on FGF2 signaling. A combination of Wnt and FGF signaling may provide a therapeutic strategy for in vitro expansion or in vivo activation of adult retinal stem cells.

Serum amyloid A (SAA) is a sensitive marker of acute-phase responses and known as a precursor protein of amyloid fibril in amyloid A (AA) (secondary) amyloidosis. Since the serum SAA level is also closely related to activity of chronic inflammatory disease and coronary artery disease, it is important to clarify the exact induction mechanism of SAA from the clinical point of view. Here we provide evidence that STAT3 plays an essential role in cytokine-driven SAA expression, although the human SAA gene shows no typical STAT3 response element (RE) in its promoters. STAT3 and nuclear factor kappaB (NF-kappaB) p65 first form a complex following interleukin (IL)-1 and IL-6 (IL-1+6) stimulation, after which STAT3 interacts with nonconsensus sequences at a 3' site of the SAA gene promoter's NF-kappaB RE. Moreover, co-expression of p300 with STAT3 dramatically enhances the transcriptional activity of SAA. The formation of a complex with STAT3, NF-kappaB p65, and p300 is thus essential for the synergistic induction of the SAA gene by IL-1+6 stimulation. Our findings are expected to aid the understanding of the inflammatory status of AA amyloidosis to aid development of a therapeutic strategy for this disease by means of normalization of serum SAA levels.

Neuronal and glial cells organizing the central nervous system (CNS) are generated from common neural precursor cells (NPCs) during neural development. However, the expression of cell-surface glycoconjugates that are crucial for determining the properties and biological function of these cells at different stages of development has not been clearly defined. In this study, we investigated the expression of several stage-specific glycoconjugate antigens, including several b-series gangliosides GD3, 9-O-acetyl GD3, GT1b and GQ1b, stage-specific embryonic antigen-1 (SSEA-1) and HNK-1, in mouse embryonic NPCs employing immunocytochemistry and flow cytometry. In addition, several of these antigens were positively identified by chemical means for the first time. We further showed that the SSEA-1 immunoreactivity was contributed by both glycoprotein and glycolipid antigens, and that of HNK-1 was contributed only by glycoproteins. Functionally, SSEA-1 may participate in migration of the cells from neurospheres in an NPC cell culture system, and the effect could be repressed by anti-SSEA-1 antibody. Based on this observation, we identified β1 integrin as one of the SSEA-1 carrier glycoproteins. Our data thus provide insights into the functional role of certain glycoconjugate antigens in NPCs during neural development.

The purpose of this study was to investigate the differentiation of neural progenitor cells (NPCs) following retinal transplantation in N -methyl- d -aspartate (NMDA)-treated eyes. NMDA was injected into the vitreous cavity of adult rat eyes. NPCs were prepared from telencephalic neuroepithelium of enhanced green fluorescence protein (EGFP) transgenic mice on embryonic day 14.5. A cell suspension was injected into the vitreous cavity in experimental eyes. Immunohistochemistry was conducted at 1, 2 or 4 weeks after transplantation of NPCs in an effort to determine the survival and differentiation of transplanted NPCs. Similar experiments were conducted using glycoprotein (gp)130-null (−/−) mice. Examination of retinal sections revealed that transplanted NPCs could survive for at least 4 weeks in NMDA-treated retinas. Immunohistochemical studies for specific cell-type markers revealed that, among the transplanted NPCs at 2 weeks after transplantation, the mean percentage (±standard deviation) of glial fibrillary acidic protein (GFAP)-positive (glial) cells was 63.5 ± 7.4%, demonstrating the differentiation of transplanted NPCs with a preference for the glial lineage. Furthermore, the mean percentage of βIII-tubulin-positive (mature neuronal) cells was 18.8 ± 4.5%. Following transplantation of NPCs isolated from gp130−/− mice into NMDA-treated retinas, the mean percentage of GFAP-positive cells (17.6 ± 7.0%), was significantly lower than that in NPCs isolated from wild-type mice (59.1 ± 6.0%, P = 0.04, Mann–Whitney U test). Preferential differentiation of NPCs into the glial lineage is induced through gp130 signaling in NMDA-treated eyes.

Activation of the transcription factor STAT3 is important for astrocyte differentiation during neural development. Demethylation of the methyl-CpG dinucleotide in the STAT3 binding site in the promoter of the glial fibrillary acidic protein (GFAP) gene, a marker for astrocytes, was previously shown to be a crucial cue for neural progenitors to express this gene in response to astrogenic signals during brain development. In this study, we analyzed the methylation status of the STAT3 binding site in the GFAP gene promoter during neural cell development from mouse embryonic stem (ES) cells in vitro. The CpG dinucleotide in the STAT3 binding site in the GFAP gene promoter exhibited a high incidence of cytidine-methylation in undifferentiated pluripotent ES cells. The high incidence of methylation of this particular cytidine was maintained in ES cell-derived neuroectoderm-like cells, but it underwent demethylation when the neural lineage cells became competent to express GFAP in response to a STAT3 activation signal. In contrast, hypermethylation of the CpG site was maintained in non-neural cells generated from the same ES cells. Progressive demethylation of the STAT3 binding site in the GFAP gene promoter was also observed in primary embryonic neuroepithelial cells during in vitro culture, whereas non-neural cells maintained hypermethylation of this site even after culture. Taken together, these results demonstrate that the astrocyte gene-specific cytidine-demethylation is programmed when neural progenitors from pluripotent cells are committed to a neural lineage that is capable of producing astrocytes.

Three principal cell types in the central nervous system, i.e., neurons, astrocytes and oligodendrocytes, arise from neural stem cells. Recent studies have revealed that such stem cells are present in the adult brain as well as in the developing brain. Recent advances enabled us to understand molecular regulation of neural stem cells, raising a possibility that they can be used for therapeutic treatment of neurodegenerative diseases. Differentiation of neural stem cells into the three neural cell types is known to be regulated in part by cell-external cues. For example, cytokines in the interleukin-6 (IL-6) family and the transforming growth factor β (TGF β) family promote astrocytogenesis. In the nucleus of cytokine-treated cells, multiple physical interactions among transcription factors and transcriptional co-activators occur and contribute to the neuroglial differentiation. Upon injury, cytokines secreted from injured tissues affect cell fates of neighboring stem cells during nerve regeneration in the adult. In this case, undesired gliosis is often observed, which is most likely induced by such cytokines. In addition to cell-external cues, cell fate determination process is also regulated by cell-intrinsic programs, which include epigenetic regulation. Spationtemporally regulated expression of lineage specific genes are important for fate determination of neural stem cells, which in some cases is associated with DNA methylation and histone modification within their promoters. From these viewpoints, we here discuss molecular regulation of neural stem cells during development and regeneration. Efforts to apply our findings to therapeutic treatment of neurodegenerative disorders are also discussed.

Spontaneous recovery after spinal cord injury is limited. Transplantation of neural precursor cells (NPCs) into lesioned adult rat spinal cord results in only partial functional recovery, and most transplanted cells tend to differentiate predominantly into astrocytes. In order to improve functional recovery after transplantation, it is important that transplanted neural precursor cells appropriately differentiate into cell lineages required for spinal cord regeneration. In order to modulate the fate of transplanted cells, we advocate transplanting gene-modified neural precursor cells. We demonstrate that gene modification to inhibit bone morphogenetic protein (BMP) signaling by noggin expression promoted differentiation of neural precursor cells into neurons and oligodendrocytes, in addition to astrocytes after transplantation. Furthermore, functional recovery of the recipient mice with spinal cord injury was observed when noggin-expressing neural precursor cells were transplanted. These observations suggest that gene-modified neural precursor cells that express molecules involved in cell fate modulation could improve central nervous system (CNS) regeneration.

Neurons, astrocytes and oligodendrocytes arise from common progenitor cells that reside in the neuroepithelium of the developing brain. We have focused on cytokines as astrocyte-inducing cell external cues, and found that leukemia inhibitory factor (LIF) and bone morphogenetic protein 2 (BMP2) synergistically induce glial fibrillary acidic protein (GFAP)-positive astrocytes. The synergistic action of these two cytokines is achieved by the complex formation between respective downstream transcription factors, STAT3 and Smad1, bridged by a transcriptional coactivator p300. Simultaneously, BMP2 also represses the differentiation of neurons by inducing anti-neurogenic basic helix-loop-helix (bHLH) transcription factors, Id1, Id3 and Hes-5. Astrocyte-inducing cytokines not only promote astrocytogenesis but also inhibit neurogenesis, suggesting the presence of negative interaction between neuronal and astrocytic differentiation pathways. We also found that oligodendrocytic bHLH factor OLIG2 inhibits the LIF-induced transcription of GFAP gene by abolishing the complex formation between STAT3 and p300. This suggests the presence of negative interaction between astrocytic and oligodendrocytic cell lineages, same as has been observed for neuronal and astrocytic cell lineages. Interestingly, differentiation of neuroepithelial cells depends on cell intrinsic programs, in addition to cell external cues. In contrast to embryonic day 14.5 (E14.5) neuroepithelial cells, LIF is not sufficient to induce GFAP expression in E11.5 neuroepithelial cells. Analysis of the GFAP gene promoter revealed that CpG dinucleotide sequences within the STAT3 recognition site is methylated in E11.5 neuroepithelial cells, and this methylation leads to the inaccessibility of activated STAT3 to its binding elements. The methylation frequency in the STAT3 binding site in the GFAP promoter declines as the developmental process proceeds, allowing the expression of GFAP in astrocytic cell lineage. The results suggest that lineage specification is regulated by cell-external cues and cell-intrinsic programs, where the former involves extracellular cytokines and the latter includes DNA methylation of cell lineage specific gene promoters. Our work also suggests the presence of negative regulatory interactions among the signals that promote differentiation of neurons, astrocytes and oligodendrocytes.

The t(8;13) translocation, found in a rare and aggressive type of stem cell myeloproliferative disorder, leads to the generation of a fusion protein between the N-terminal gene product of fused in myeloproliferative disorders (FIM)/ZNF198 and the fibroblast growth factor receptor 1 (FGFR1) kinase domain. The chimeric protein was reported to have constitutively activated tyrosine kinase activity. However, little is known about a role of FIM in hematopoietic cell regulation. Here we show that FIM protein is ubiquitously expressed in mouse embryonic tissues but much less in hematopoietic cells. We also show that forced expression of FIM inhibits the emergence of hematopoietic cells in the cultured mouse aorta-gonad-mesonephros (AGM) region on embryonic day (E) 11.5, where definitive hematopoiesis is first found during embryogenesis. These results suggest that the expression level of FIM determines the development of hematopoiesis during mouse ontogeny.

In midgestation mouse embryos, the aorta-gonad-mesonephros (AGM) region generates hematopoietic stem cells and definitive hematopoiesis is regulated by cell–cell interaction and signaling molecules. We showed that a Ras/mitogen-activated protein (MAP) kinase signaling-specific inhibitor and a dominant negative mutant Ras blocked the production of CD45+ hematopoietic cells in embryonic day 11.5 AGM culture, indicating an essential role for the MAP kinase pathway in AGM hematopoiesis. Overexpression of the Ras/MAP kinase pathway regulator, Spred-2, in the AGM culture significantly reduced the number of CD45+ cells. In contrast, production of CD45+ cells from the AGM region of Spred-2–null mice was up-regulated as compared with wild-type littermates. Furthermore, Spred-2–deficient mice exhibited elevated hematopoietic colony formation from vascular endothelial-cadherin+ cells. These data indicate that Spred-2 functions as a negative regulator of AGM hematopoiesis by inhibiting hematopoietic cytokine signaling.